Science.gov

Sample records for chinese bombyx mandarina

  1. Transcriptome Sequencing and Positive Selected Genes Analysis of Bombyx mandarina

    PubMed Central

    Wu, Yuqian; Long, Renwen; Liu, Chun; Xia, Qingyou

    2015-01-01

    The wild silkworm Bombyx mandarina is widely believed to be an ancestor of the domesticated silkworm, Bombyx mori. Silkworms are often used as a model for studying the mechanism of species domestication. Here, we performed transcriptome sequencing of the wild silkworm using an Illumina HiSeq2000 platform. We produced 100,004,078 high-quality reads and assembled them into 50,773 contigs with an N50 length of 1764 bp and a mean length of 941.62 bp. A total of 33,759 unigenes were identified, with 12,805 annotated in the Nr database, 8273 in the Pfam database, and 9093 in the Swiss-Prot database. Expression profile analysis found significant differential expression of 1308 unigenes between the middle silk gland (MSG) and posterior silk gland (PSG). Three sericin genes (sericin 1, sericin 2, and sericin 3) were expressed specifically in the MSG and three fibroin genes (fibroin-H, fibroin-L, and fibroin/P25) were expressed specifically in the PSG. In addition, 32,297 Single-nucleotide polymorphisms (SNPs) and 361 insertion-deletions (INDELs) were detected. Comparison with the domesticated silkworm p50/Dazao identified 5,295 orthologous genes, among which 400 might have experienced or to be experiencing positive selection by Ka/Ks analysis. These data and analyses presented here provide insights into silkworm domestication and an invaluable resource for wild silkworm genomics research. PMID:25806526

  2. A Bombyx mandarina mutant exhibiting translucent larval skin is controlled by the molybdenum cofactor sulfurase gene.

    PubMed

    Fujii, Tsuguru; Ozaki, Masataka; Masamoto, Takaaki; Katsuma, Susumu; Abe, Hiroaki; Shimada, Toru

    2009-04-01

    During the maintenance of the wild silkworm, Bombyx mandarina, a mutant phenotype exhibiting translucent skin was identified. Based on the crossing experiments with the domesticated silkworm, Bombyx mori, we found that the mutant was controlled by molybdenum cofactor sulfurase (MoCoS) gene. We designated the mutant ''Ozaki's translucent'' (og(Z)). We found a 2.1-kb deletion containing the transcription initiation site, exons 1 and 2, and the 5' end of exon 3 of the MoCoS gene. The transcript of the MoCoS gene was not detected in the og(Z) homozygote. We concluded that og(Z) is a complete loss-of-function allele generated by a disruption of the MoCoS gene.

  3. IDENTIFICATION AND EXPRESSION ANALYSIS OF VITELLOGENIN RECEPTOR FROM THE WILD SILKWORM, Bombyx mandarina.

    PubMed

    Qian, Cen; Fu, Wei-Wei; Wei, Guo-Qing; Wang, Lei; Liu, Qiu-Ning; Dai, Li-Shang; Sun, Yu; Zhu, Bao-Jian; Liu, Chao-Liang

    2015-08-01

    The vitellogenin receptor (VgR) plays a key role on embryonic development in oviparous animals. Here, we cloned a VgR gene, which was identified from the wild silkworm Bombyx mandarina (BmaVgR) using reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Sequence analysis revealed that BmaVgR is 5,861 bp long with an open reading frame encoded by 1,811 amino acid residues. The predicted amino acid sequence has 99.7 and 98.2% identity with the VgRs of Actias selene and Bombyx mori, respectively. The class B domain sequence of BmaVgR was cloned and expressed in Escherichia coli, and purified by a Ni-NTA column. Polyclonal antibodies were produced against the purified recombinant protein, and titer of the antibody was about 1:12,800 measured by enzyme-linked immunosorbent assay (ELISA). Western blot and RT-qPCR showed that BmaVgR was expressed in the ovary and fat body of female larvae and the ovary of moth, and the expression level was highest at the third day and then declined from third day to seventh in fat body of pupa. After knockdown of the BmaVgR gene through RNA interference (RNAi), other three BmaVgR-related genes (Vg, egg-specific protein, and low molecular weight lipoprotein LP gene) were all downregulated significantly. © 2015 Wiley Periodicals, Inc.

  4. Two novel Pao-like retrotransposons (Kamikaze and Yamato) from the silkworm species Bombyx mori and B. mandarina: common structural features of Pao-like elements.

    PubMed

    Abe, H; Ohbayashi, F; Sugasaki, T; Kanehara, M; Terada, T; Shimada, T; Kawai, S; Mita, K; Kanamori, Y; Yamamoto, M T; Oshiki, T

    2001-04-01

    To characterize the structural features common to Pao-like retrotransposons, we analyzed two lambda phage clones which contain the Pao-like elements from the silkworm species Bombyx mori and B. mandarinia, and copies of Pao itself and ninja of Drosophila simulans, amplified by PCR. We previously identified two randomly amplified polymorphic DNAs (RAPDs), W-Kamikaze and W-Yamato, from B. mori and B. mandarina, which are part of two novel Pao-like retrotransposons, Kamikaze and Yamato, respectively. Complete characterization of these and other elements of this group reported here shows that Pao-like elements have common features that distinguish them from the other groups of LTR-retrotransposons. While the elements of the Ty1-copia group encode only one cysteine and histidine (Cys) motif in their gag-like region, the Pao-like elements specify three Cys motifs. The highly conserved D(35)E motif in the integrase domain of the retrotransposon polyprotein seems to be conserved in Pao-like elements, but the number of amino acid residues between D and E varies and is greater than 35. A comparison of the deduced amino acid sequences of the reverse transcriptase domain revealed the Pao-like elements are members of neither the Ty1-copia nor the gypsy-Ty3 groups. Therefore, we confirmed that the long-terminal-repeat (LTR) retrotransposons should be divided into three major groups (or families), namely the Ty1-copia, gypsy-Ty3, and Pao-like groups.

  5. Identification of novel random amplified polymorphic DNAs (RAPDs) on the W chromosome of the domesticated silkworm, Bombyx mori, and the wild silkworm, B. mandarina, and their retrotransposable element-related nucleotide sequences.

    PubMed

    Abe, H; Kanehara, M; Terada, T; Ohbayashi, F; Shimada, T; Kawai, S; Suzuki, M; Sugasaki, T; Oshiki, T

    1998-08-01

    Genomic DNAs were compared between males and females of the domesticated silkworm, Bombyx mori, strains C108, C137, J137, p50, and WILD-W (constructed by crossing a wild silkworm, B. mandarina, female with a male of strain C108) by polymerase chain reaction (PCR) with 700 arbitrary 10-mer primers. Four female-specific RAPDs (W-Kabuki, W-Samurai, W-Kamikaze, and W-Yamato) were found. The sex chromosome formulas of B. mori and B. mandarina are ZW (XY) for the female and ZZ (XX) for the male. The four female-specific RAPDs are assumed to be derived from the W chromosome because the other chromosomes are shared by both sexes. A computer search for deduced amino acid sequences of these four RAPDs revealed that all of them showed homology to previously reported amino acid sequences encoded in known retrotransposable elements from various organisms.

  6. D-Pinitol in Fabaceae: an Oviposition Stimulant for the Common Grass Yellow Butterfly, Eurema mandarina.

    PubMed

    Mukae, Shin-Ya; Ohashi, Toshiki; Matsumoto, Yuika; Ohta, Shinji; Ômura, Hisashi

    2016-11-01

    The common grass yellow butterfly, Eurema mandarina (formerly Eurema hecabe mandarina) (Lepidoptera, Pieridae), recently has been separated taxonomically from a subtropical population of Eurema hecabe in Japan. This species is widely distributed in the temperate region of Japan, and feeds mainly on various ligneous plants within the Fabaceae. We attempted to identify an oviposition stimulant for E. mandarina from its primary hosts, Albizia julibrissin and Lespedeza cuneata. In both hosts, crude extract and an aqueous fraction elicited oviposition responses from gravid females. A polar subfraction of the aqueous fraction also stimulated high oviposition-stimulatory activity, comparable to the original aqueous fraction, suggesting that E. mandarina females use water-soluble compounds for host recognition. Subsequent activity-directed fractionation by ion exchange chromatography indicated that one of the key substances was contained in the neutral/amphoteric fraction. Chemical analyses revealed that the active fractions of both hosts contained D-(+)-pinitol as the major component. We examined female responses to authentic D-pinitol and found that it induced oviposition responses at concentrations greater than 0.1 %. Since this cyclitol is omnipresent in Fabaceae, we conclude that D-pinitol plays a role in mediating oviposition of E. mandarina on fabaceous plants.

  7. Anatomical and functional analysis of domestication effects on the olfactory system of the silkmoth Bombyx mori

    PubMed Central

    Bisch-Knaden, Sonja; Daimon, Takaaki; Shimada, Toru; Hansson, Bill S.; Sachse, Silke

    2014-01-01

    The silkmoth Bombyx mori is the main producer of silk worldwide and has furthermore become a model organism in biological research, especially concerning chemical communication. However, the impact domestication might have had on the silkmoth's olfactory sense has not yet been investigated. Here, we show that the pheromone detection system in B. mori males when compared with their wild ancestors Bombyx mandarina seems to have been preserved, while the perception of environmental odorants in both sexes of domesticated silkmoths has been degraded. In females, this physiological impairment was mirrored by a clear reduction in olfactory sensillum numbers. Neurophysiological experiments with hybrids between wild and domesticated silkmoths suggest that the female W sex chromosome, so far known to have the sole function of determining femaleness, might be involved in the detection of environmental odorants. Moreover, the coding of odorants in the brain, which is usually similar among closely related moths, differs strikingly between B. mori and B. mandarina females. These results indicate that domestication has had a strong impact on odour detection and processing in the olfactory model species B. mori. PMID:24258720

  8. Anatomical and functional analysis of domestication effects on the olfactory system of the silkmoth Bombyx mori.

    PubMed

    Bisch-Knaden, Sonja; Daimon, Takaaki; Shimada, Toru; Hansson, Bill S; Sachse, Silke

    2014-01-07

    The silkmoth Bombyx mori is the main producer of silk worldwide and has furthermore become a model organism in biological research, especially concerning chemical communication. However, the impact domestication might have had on the silkmoth's olfactory sense has not yet been investigated. Here, we show that the pheromone detection system in B. mori males when compared with their wild ancestors Bombyx mandarina seems to have been preserved, while the perception of environmental odorants in both sexes of domesticated silkmoths has been degraded. In females, this physiological impairment was mirrored by a clear reduction in olfactory sensillum numbers. Neurophysiological experiments with hybrids between wild and domesticated silkmoths suggest that the female W sex chromosome, so far known to have the sole function of determining femaleness, might be involved in the detection of environmental odorants. Moreover, the coding of odorants in the brain, which is usually similar among closely related moths, differs strikingly between B. mori and B. mandarina females. These results indicate that domestication has had a strong impact on odour detection and processing in the olfactory model species B. mori.

  9. [Scientific connotation of processing Bombyx Batryticatus under high temperature].

    PubMed

    Ma, Li; Wang, Xuan; Ma, Lin; Wang, Man-yuan; Qiu, Feng

    2015-12-01

    The aim of this study was to elucidate the scientific connotation of Bombyx Batryticatus processing with wheat bran under high temperature. The contents of soluble protein extracted from Bombyx Batryticatus and its processed products and the limited content of AFT in Bombyx Batryticatus and the processed one were compared. The concentration of protein was measured with the Bradford methods and the difference of protein between Bombyx Batryticatus and its processed products was compared by SDS-PAGE analysis. Aflatoxin B1, B2, G1, and G2 were determined by reversed-phase HPLC. The results showed that the soluble protein content of Bombyx Batryticatus and its processed products were (47.065 +/- 0.249), (29.756 +/- 1.961) mg x g(-1), correspondingly. Analysis of protein gel electrophoresis showed that there were no significant differences between the crude and processed one in protein varieties. 6 bands were detected: 31.90, 26.80, 18.71, 15.00, 10.18, 8.929 kDa. Below 10 kDa, the color of bands of the processed one was deeper than the crude one, which demonstrate that macromolecular protein was degradated into micromolecule. The content of AFG1, AFB1, AFG2, AFB2 were 0.382, 0.207, 0.223, 0.073 g x kg(-1), not exceeded 5 microg x kg(-1) while the processed one was not detected. Through processing with wheat bran under high temperature, the content of soluble protein in Bombyx Batryticatus decreased, the processing purpose for alleviating drug property was achieved. Meanwhile, the limited content of aflatoxins were reduced or cleared by processing procedure or absorbed by processing auxillary material, adding the safety of the traditional Chinese Medicine. In conclusion, as a traditional processing method, bran frying Bombyx Batryticatus was scientific and reasonable.

  10. Survey and Analysis of Microsatellites in the Silkworm, Bombyx mori

    PubMed Central

    Prasad, M. Dharma; Muthulakshmi, M.; Madhu, M.; Archak, Sunil; Mita, K.; Nagaraju, J.

    2005-01-01

    We studied microsatellite frequency and distribution in 21.76-Mb random genomic sequences, 0.67-Mb BAC sequences from the Z chromosome, and 6.3-Mb EST sequences of Bombyx mori. We mined microsatellites of ≥15 bases of mononucleotide repeats and ≥5 repeat units of other classes of repeats. We estimated that microsatellites account for 0.31% of the genome of B. mori. Microsatellite tracts of A, AT, and ATT were the most abundant whereas their number drastically decreased as the length of the repeat motif increased. In general, tri- and hexanucleotide repeats were overrepresented in the transcribed sequences except TAA, GTA, and TGA, which were in excess in genomic sequences. The Z chromosome sequences contained shorter repeat types than the rest of the chromosomes in addition to a higher abundance of AT-rich repeats. Our results showed that base composition of the flanking sequence has an influence on the origin and evolution of microsatellites. Transitions/transversions were high in microsatellites of ESTs, whereas the genomic sequence had an equal number of substitutions and indels. The average heterozygosity value for 23 polymorphic microsatellite loci surveyed in 13 diverse silkmoth strains having 2–14 alleles was 0.54. Only 36 (18.2%) of 198 microsatellite loci were polymorphic between the two divergent silkworm populations and 10 (5%) loci revealed null alleles. The microsatellite map generated using these polymorphic markers resulted in 8 linkage groups. B. mori microsatellite loci were the most conserved in its immediate ancestor, B. mandarina, followed by the wild saturniid silkmoth, Antheraea assama. PMID:15371363

  11. Expression in Escherichia coli and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silk worm, Bombyx mori.

    PubMed

    Li, Bao-Cun; Zhang, Shuang-Quan; Dan, Wen-Bing; Chen, Yu-Qing; Cao, Peng

    2007-07-01

    The antibacterial peptide CM4 (ABP-CM4), isolated from Chinese Bombys mori, is a 35-residue cationic, amphipathic alpha-helical peptide that exhibits a broad range of antimicrobial activity. To explore a new approach for the expression of ABP-CM4 in E. coli, the gene ABP-CM4, obtained by recursive PCR (rPCR), was cloned into the vector pET32a to construct a fusion expression plasmid. The fusion protein Trx-CM4 was expressed in soluble form, purified by Ni(2+)-chelating chromatography, and cleaved by formic acid to release recombinant CM4. Purification of rCM4 was achieved by affinity chromatography and reverse-phase HPLC. The purified of recombinant peptide showed antimicrobial activities against E. coli K(12)D(31), Penicillium chrysogenum, Aspergillus niger and Gibberella saubinetii. According to the antimicrobial peptide database (http://aps.unmc.edu/AP/main.html), 116 peptides contain a Met residue, but only 5 peptides contain the AspPro site, indicating a broader application of formic acid than CNBr in cleaving fusion protein. The successful application to the expression of the ABP-CM4 indicates that the system is a low-cost, efficient way of producting milligram quantities of ABP-CM4 that is biologically active.

  12. Contrasting response to Pleistocene climate change by ground-living and arboreal Mandarina snails from the oceanic Hahajima archipelago.

    PubMed

    Davison, Angus; Chiba, Satoshi

    2008-10-27

    While the genetic impact of Pleistocene climate change on temperate species has been well characterized, especially in Europe and North America, an effect on the diversification of species on oceanic islands has been less well studied. This is perhaps a surprising observation given the traditional and continuing contribution of island species (e.g. Darwin's finches, Partula snails, Lord Howe Island palms) to understand speciation. Here, we combine mitochondrial and microsatellite data from the ground-living and arboreal Mandarina snails of the oceanic, subtropical Hahajima archipelago (Ogasawara, colloquially 'Galápagos of the Orient') to enable a comparative approach to understand the impact of the Pleistocene glaciations on their phylogeography. Prior work suggested that several narrowly divergent, ground-living species pairs of Mandarina populations on the outlying islands, as well as the low-lying southern and central parts of Hahajima, probably underwent bottlenecks and subsequent expansions during the recent Pleistocene. Here, the most striking finding is that largely arboreal species have deeply divergent, geographically restricted mitochondrial lineages, in contrast to a census size that is at least an order of magnitude lower than ground-living snails. As populations of both types are highly polymorphic at microsatellite loci, the systematic difference at the mitochondrial locus probably indicates a contrasting effect of the Pleistocene climate cycles on the two groups. We speculate that this may have partly come about owing to a reduced efficacy of natural selection on the more greatly structured populations of arboreal snails. If so, then a prediction is that the genome of other snails, or other species with limited mobility, will show a similar response to the Pleistocene climate cycles.

  13. A Hox Gene, Antennapedia, Regulates Expression of Multiple Major Silk Protein Genes in the Silkworm Bombyx mori*

    PubMed Central

    Tsubota, Takuya; Tomita, Shuichiro; Uchino, Keiro; Kimoto, Mai; Takiya, Shigeharu; Kajiwara, Hideyuki; Yamazaki, Toshimasa; Sezutsu, Hideki

    2016-01-01

    Hox genes play a pivotal role in the determination of anteroposterior axis specificity during bilaterian animal development. They do so by acting as a master control and regulating the expression of genes important for development. Recently, however, we showed that Hox genes can also function in terminally differentiated tissue of the lepidopteran Bombyx mori. In this species, Antennapedia (Antp) regulates expression of sericin-1, a major silk protein gene, in the silk gland. Here, we investigated whether Antp can regulate expression of multiple genes in this tissue. By means of proteomic, RT-PCR, and in situ hybridization analyses, we demonstrate that misexpression of Antp in the posterior silk gland induced ectopic expression of major silk protein genes such as sericin-3, fhxh4, and fhxh5. These genes are normally expressed specifically in the middle silk gland as is Antp. Therefore, the evidence strongly suggests that Antp activates these silk protein genes in the middle silk gland. The putative sericin-1 activator complex (middle silk gland-intermolt-specific complex) can bind to the upstream regions of these genes, suggesting that Antp directly activates their expression. We also found that the pattern of gene expression was well conserved between B. mori and the wild species Bombyx mandarina, indicating that the gene regulation mechanism identified here is an evolutionarily conserved mechanism and not an artifact of the domestication of B. mori. We suggest that Hox genes have a role as a master control in terminally differentiated tissues, possibly acting as a primary regulator for a range of physiological processes. PMID:26814126

  14. A Hox Gene, Antennapedia, Regulates Expression of Multiple Major Silk Protein Genes in the Silkworm Bombyx mori.

    PubMed

    Tsubota, Takuya; Tomita, Shuichiro; Uchino, Keiro; Kimoto, Mai; Takiya, Shigeharu; Kajiwara, Hideyuki; Yamazaki, Toshimasa; Sezutsu, Hideki

    2016-03-25

    Hoxgenes play a pivotal role in the determination of anteroposterior axis specificity during bilaterian animal development. They do so by acting as a master control and regulating the expression of genes important for development. Recently, however, we showed that Hoxgenes can also function in terminally differentiated tissue of the lepidopteranBombyx mori In this species,Antennapedia(Antp) regulates expression of sericin-1, a major silk protein gene, in the silk gland. Here, we investigated whether Antpcan regulate expression of multiple genes in this tissue. By means of proteomic, RT-PCR, and in situ hybridization analyses, we demonstrate that misexpression of Antpin the posterior silk gland induced ectopic expression of major silk protein genes such assericin-3,fhxh4, and fhxh5 These genes are normally expressed specifically in the middle silk gland as is Antp Therefore, the evidence strongly suggests that Antpactivates these silk protein genes in the middle silk gland. The putativesericin-1 activator complex (middle silk gland-intermolt-specific complex) can bind to the upstream regions of these genes, suggesting that Antpdirectly activates their expression. We also found that the pattern of gene expression was well conserved between B. moriand the wild species Bombyx mandarina, indicating that the gene regulation mechanism identified here is an evolutionarily conserved mechanism and not an artifact of the domestication of B. mori We suggest that Hoxgenes have a role as a master control in terminally differentiated tissues, possibly acting as a primary regulator for a range of physiological processes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Bombyx small RNAs: genomic defense system against transposons in the silkworm, Bombyx mori.

    PubMed

    Kawaoka, Shinpei; Hayashi, Nobumitsu; Katsuma, Susumu; Kishino, Hirohisa; Kohara, Yuji; Mita, Kazuei; Shimada, Toru

    2008-12-01

    Selfish genetic elements called transposons can insert themselves at new locations in host genomes to modify gene structure and alter gene expression. Expansion of transposons can occur when novel transposition events are transmitted to subsequent generations after germline hopping. Therefore, organisms seem likely to have evolved defense mechanisms to silence transposons in the germline. Recently, small RNAs interacting with Piwi proteins (piwi-interacting RNAs: piRNAs) have been demonstrated to be involved in genomic defense mechanism against transposons. Here, we show that piRNA-like small RNAs are present abundantly in the Bombyx ovary. We cloned 38,493 kinds of Bombyx small RNA from the ovary and performed functional characterization. Bombyx small RNAs showed a unimodal length distribution with a peak at 28nt and a strong bias for U at the 5' end. We found that 12,869 kinds of Bombyx small RNAs were associated with transposons or repetitive sequences. We classified them as repeat-associated small interfering RNAs (rasiRNAs), a subclass of piRNAs. Notably, antisense rasiRNAs have a strong bias toward U at 5' ends; in contrast, sense rasiRNAs have a strong bias toward A at nucleotide position 10, indicating that the piRNA amplification loop proposed in Drosophila is evolutionarily conserved in Bombyx. These results suggest that Bombyx small RNAs regulate transposon activity.

  16. Bombyx mori pylorus infection by Alphabaculovirus.

    PubMed

    Baggio, M P D; Ribeiro, L F C; Vessaro-Silva, S A; Brancalhão, R M C

    2014-04-03

    Alphabaculovirus is an entomopathogenic virus genus that infects Bombyx mori, which is known as the Bombyx mori multiple nucleopolyedrovirus (BmMNPV). This virus is polyorganotrophic, and a series of tissues are known as targets; however, there is currently no information regarding infection in the pylorus, the segment of the hindgut that is present in the midgut transition and is responsible for food passage control. Thus, in the present study, we aimed to analyze infection of the B. mori pylorus by BmMNPV. To do so, hybrid B. mori larvae were inoculated with a viral suspension of BmMNPV, and segments of the intestine containing the pylorus and its subdivisions, the posterior interstitial ring (PIR), pyloric cone, and pyloric valve, were dissected and processed for light microscopy on different days post inoculation. The results showed that B. mori pylorus subdivisions respond differently to infection, and the anterior area of the PIR is susceptible with these cells being the secondary infection targets. Cytological analysis revealed the presence of viroplasm in the hypertrophic nucleus, followed by the formation and development of viral polyhedra. Cytolysis occurred at the end of the infectious cycle, thereby releasing polyhedra and enabling the spread of the disease. There was no evidence of BmMNPV infection in the posterior area of the PIR, cone, or pyloric valve. These results will contribute to greater understanding of the virus infectious cycle, whose consequent epizootic disease can negatively impact this economically important insect that is used in silk production in Brazil.

  17. Materials fabrication from Bombyx mori silk fibroin.

    PubMed

    Rockwood, Danielle N; Preda, Rucsanda C; Yücel, Tuna; Wang, Xiaoqin; Lovett, Michael L; Kaplan, David L

    2011-09-22

    Silk fibroin, derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has controllable degradation rates from hours to years and can be chemically modified to alter surface properties or to immobilize growth factors. A variety of aqueous or organic solvent-processing methods can be used to generate silk biomaterials for a range of applications. In this protocol, we include methods to extract silk from B. mori cocoons to fabricate hydrogels, tubes, sponges, composites, fibers, microspheres and thin films. These materials can be used directly as biomaterials for implants, as scaffolding in tissue engineering and in vitro disease models, as well as for drug delivery.

  18. Materials Fabrication from Bombyx mori Silk Fibroin

    PubMed Central

    Rockwood, Danielle N.; Preda, Rucsanda C.; Yücel, Tuna; Wang, Xiaoqin; Lovett, Michael L.; Kaplan, David L.

    2013-01-01

    Silk fibroin, derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has controllable degradation rates from hours to years, and it can be chemically modified to alter surface properties or to immobilize growth factors. A variety of aqueous or organic solvent processing methods can be used to generate silk biomaterials for a range of applications. In this protocol we include methods to extract silk from B. mori cocoons in order to fabricate hydrogels, tubes, sponges, composites, fibers, microspheres and thin films. These materials can be used directly as biomaterials for implants, as scaffolding in tissue engineering and in vitro disease models, and for drug delivery. PMID:21959241

  19. Chondroitinase from baculovirus Bombyx mori nucleopolyhedrovirus and chondroitin sulfate from silkworm Bombyx mori.

    PubMed

    Sugiura, Nobuo; Ikeda, Motoko; Shioiri, Tatsumasa; Yoshimura, Mayumi; Kobayashi, Michihiro; Watanabe, Hideto

    2013-12-01

    Chondroitin sulfate (CS) is a linear polysaccharide composed of repeating disaccharide units of glucuronic acid (GlcUA) and N-acetyl-d-galactosamine (GalNAc) with sulfate groups at various positions. Baculovirus is an insect-pathogenic virus that infects Lepidoptera larvae. Recently, we found that the occlusion-derived virus envelope protein 66 (ODV-E66) from Autographa californica nucleopolyhedrovirus (AcMNPV) exhibits chondroitin (CH)-digesting activity with distinct substrate specificity. Here, we demonstrate that the ODV-E66 protein from Bombyx mori nucleopolyhedrovirus (BmNPV) exhibits 92% homology to the amino acid sequence and 83% of the CH lyase activity of ODV-E66 from AcMNPV. ODV-E66 cleaves glycosyl bonds at nonreducing sides of disaccharide units consisting of nonsulfated and 6-O-sulfated GalNAc residues. We then investigated CS in the silkworm, Bombyx mori, which is the host of BmNPV. CS was present in insect tissues such as the midgut, peritrophic membrane, silk gland and skin. The polysaccharide consisted of a nonsulfated disaccharide unit, mono-sulfated disaccharide at Position 4 of the GalNAc residue and mono-sulfated disaccharide at Position 6 of the GalNAc residue. With regard to immunohistochemical analysis, the staining patterns of the silkworm tissues were different among anti-CS antibodies. Chondroitn sulfate that is digestible by ODV-E66 exists sufficiently in the peritrophic membrane protecting the midgut epithelium from ingested pathogens. Our results suggest that ODV-E66 facilitates the primary infection of the virus by digestion of CS in the peritrophic membrane.

  20. The genomic underpinnings of apoptosis in the silkworm, Bombyx mori

    PubMed Central

    2010-01-01

    Background Apoptosis is regulated in an orderly fashion by a series of genes, and has a crucial role in important physiological processes such as growth development, immunological response and so on. Recently, substantial studies have been undertaken on apoptosis in model animals including humans, fruit flies, and the nematode. However, the lack of genomic data for silkworms limits their usefulness in apoptosis studies, despite the advantages of silkworm as a representative of Lepidoptera and an effective model system. Herein we have identified apoptosis-related genes in the silkworm Bombyx mori and compared them to those from insects, mammals, and nematodes. Results From the newly assembled genome databases, a genome-wide analysis of apoptosis-related genes in Bombyx mori was performed using both nucleotide and protein Blast searches. Fifty-two apoptosis-related candidate genes were identified, including five caspase family members, two tumor necrosis factor (TNF) superfamily members, one Bcl-2 family member, four baculovirus IAP (inhibitor of apoptosis) repeat (BIR) domain family members and 1 RHG (Reaper, Hid, Grim, and Sickle; Drosophila cell death activators) family member. Moreover, we identified a new caspase family member, BmCaspase-New, two splice variants of BmDronc, and Bm3585, a mammalian TNF superfamily member homolog. Twenty-three of these apoptosis-related genes were cloned and sequenced using cDNA templates isolated from BmE-SWU1 cells. Sequence analyses revealed that these genes could have key roles in apoptosis. Conclusions Bombyx mori possesses potential apoptosis-related genes. We hypothesized that the classic intrinsic and extrinsic apoptotic pathways potentially are active in Bombyx mori. These results lay the foundation for further apoptosis-related study in Bombyx mori. PMID:21040523

  1. The genomic underpinnings of apoptosis in the silkworm, Bombyx mori.

    PubMed

    Zhang, Jin-Ye; Pan, Min-Hui; Sun, Zhi-Ya; Huang, Shu-Jing; Yu, Zi-Shu; Liu, Di; Zhao, Dan-Hong; Lu, Cheng

    2010-10-31

    Apoptosis is regulated in an orderly fashion by a series of genes, and has a crucial role in important physiological processes such as growth development, immunological response and so on. Recently, substantial studies have been undertaken on apoptosis in model animals including humans, fruit flies, and the nematode. However, the lack of genomic data for silkworms limits their usefulness in apoptosis studies, despite the advantages of silkworm as a representative of Lepidoptera and an effective model system. Herein we have identified apoptosis-related genes in the silkworm Bombyx mori and compared them to those from insects, mammals, and nematodes. From the newly assembled genome databases, a genome-wide analysis of apoptosis-related genes in Bombyx mori was performed using both nucleotide and protein Blast searches. Fifty-two apoptosis-related candidate genes were identified, including five caspase family members, two tumor necrosis factor (TNF) superfamily members, one Bcl-2 family member, four baculovirus IAP (inhibitor of apoptosis) repeat (BIR) domain family members and 1 RHG (Reaper, Hid, Grim, and Sickle; Drosophila cell death activators) family member. Moreover, we identified a new caspase family member, BmCaspase-New, two splice variants of BmDronc, and Bm3585, a mammalian TNF superfamily member homolog. Twenty-three of these apoptosis-related genes were cloned and sequenced using cDNA templates isolated from BmE-SWU1 cells. Sequence analyses revealed that these genes could have key roles in apoptosis. Bombyx mori possesses potential apoptosis-related genes. We hypothesized that the classic intrinsic and extrinsic apoptotic pathways potentially are active in Bombyx mori. These results lay the foundation for further apoptosis-related study in Bombyx mori.

  2. Identification and characterization of an arginine kinase as a major allergen from silkworm (Bombyx mori) larvae.

    PubMed

    Liu, Zhigang; Xia, Lixin; Wu, Yulan; Xia, Qingyou; Chen, Jiajie; Roux, Kenneth H

    2009-01-01

    The silkworm, Bombyx mori, is an important insect in the textile industry and its pupa are used in Chinese cuisine and traditional Chinese medicine. The silk, urine and dander of silkworms is often the cause of allergies in sericulture workers and the pupa has been found to be a food allergen in China. Recent studies have focused on reporting cases of silkworm allergies, but only a few studies have addressed the specific allergens present in the B. mori silkworm. We collected sera from 10 patients with a positive skin prick test to silkworm crude extract (SCE) and analyzed these samples by Western blot and ELISA. The cDNA of arginine kinase from the B. mori silkworm was also cloned and expressed in high yield in Escherichia coli. Allergenicity and cross-allergenicity of the recombinant B. mori arginine kinase (rBmAK) were investigated by ELISA inhibition assay. Collected sera all reacted to a 42-kDa protein in a Western blot with SCE as the antigen. Preincubation of sera with rBmAK eliminated the reactivity of the patients' sera to this 42-kDa band. All patient sera also exhibited positive reactivity to SCE in an ELISA assay. BmAK also demonstrated cross-reactivity with a recombinant AK from cockroach. Arginine kinase from the B. mori silkworm is a major allergen and crossreacts with cockroach AK. Copyright 2009 S. Karger AG, Basel.

  3. Surface display and bioactivity of Bombyx mori acetylcholinesterase on Pichia pastoris

    USDA-ARS?s Scientific Manuscript database

    To construct the Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE), the gene for the anchor protein (AGa1) was obtained from Saccharomyces cerevisiae and was fused with the modified Bombyx mori acetylcholinesterase gene (bmace) and transformed int...

  4. Characterization of the Bombyx mori Cecropin A1 promoter regulated by IMD pathway.

    PubMed

    Hua, Xiao-Ting; Ma, Xiao-Juan; Xue, Ren-Ju; Cheng, Ting-Cai; Wang, Fei; Xia, Qing-You

    2016-04-01

    Cecropin A1 (CecA1) promoter from Bombyx mori was cloned and characterized to provide insight into the transcriptional control of this antimicrobial peptide gene upon immune challenges. Reporter gene assays demonstrated that both Escherichia coli and lipopolysaccharide could induce expression in BmE cells but B. bombyseptieus or peptidoglycan failed, and the induction pattern of the reporter gene was coincident with the endogenous CecA1. Analysis of deletion and mutation constructs revealed that the regulatory region was the κB motif located between -176 and -166, and no other predicted elements on CecA1 promoter affected its inducibility. Insertion of additional κB motifs increased the activity of CecA1 promoter. Furthermore, binding of Relish to κB motif was confirmed by electrophoretic mobility shift assay. These findings indicate the regulatory mechanism of CecA1 expression in IMD pathway and suggest an approach of engineering antimicrobial peptide promoter with enhanced activities that may lead to broad applications. © 2015 Institute of Zoology, Chinese Academy of Sciences.

  5. Biological and molecular characterization of silkworm strains from the Brazilian germplasm bank of Bombyx mori.

    PubMed

    Pereira, N C; Munhoz, R E F; Bignotto, T S; Bespalhuk, R; Garay, L B; Saez, C R N; Fassina, V A; Nembri, A; Fernandez, M A

    2013-06-28

    Brazil has only one public genetic pool of Bombyx mori strains, which was established in 2005 at Universidade Estadual de Maringá, Maringá, Paraná State. This genetic bank has been maintained, and the strains have been characterized using genetic and morphological tools. The quantitative and qualitative traits, directly or indirectly related to productivity, were evaluated in 14 silkworm strains. In addition to biological and productivity analyses, DNA markers related to susceptibility to the B. mori nucleopolyhedrovirus (BmNPV) were analyzed. BmNPV is a major cause of production loss and is a serious problem for Paraná sericulture. The silkworm strains from diverse geographic origins were found to have different characteristics, including body weight, larval stage duration, cocoon weight, and other biological traits. In terms of productivity, the raw silk percentages were almost uniform, with an overall average of 16.28%. Overall, the Chinese strain C37 gave the best performance in many of the quantitative traits, and it surpassed the other strains in productivity traits. Therefore, it can be used as one of the strains that compose the elite germplasm for silkworm breeding programs. Additionally, genetic molecular markers were efficient in discriminating between B. mori strains that had been identified based on their geographical origin. We found that all Japanese strains produced a 400-bp molecular marker that has been associated with susceptibility to BmNPV.

  6. Diapause prevention effect of Bombyx mori by dimethyl sulfoxide.

    PubMed

    Yamamoto, Takayuki; Mase, Keisuke; Sawada, Hiroshi

    2013-01-01

    HCl treatment has been, for about 80 years, the primary method for the prevention of entry into embryonic diapauses of Bombyx mori. This is because no method is as effective as the HCl treatment. In this study, we discovered that dimethyl sulfoxide (DMSO) prevented entry into the diapause of the silkworm, Bombyx mori. The effect of diapause prevention was 78% as a result of treatment with 100% DMSO concentration, and the effect was comparable to that of the HCl treatment. In contrast, in the case of non-diapause eggs, hatchability was decreased by DMSO in a concentration-dependent manner. The effect of DMSO was restricted within 24 hours after oviposition of diapause eggs, and the critical period was slightly shorter than the effective period of the HCl treatment. DMSO analogs, such as dimethyl formamide (DMF) and dimethyl sulfide (DMS), did little preventive effect against the diapause. Furthermore, we also investigated the permeation effects of chemical compounds by DMSO. When treated with an inhibitor of protein kinase CK2 (CK2) dissolved in DMSO, the prevention rate of the diapause was less than 40%. This means that the inhibition effect by the CK2 inhibitor was the inhibition of embryonic development after diapause prevention by DMSO. These data suggest that DMSO has the effects of preventing from entering into the diapause and permeation of chemicals into diapause eggs.

  7. Diapause Prevention Effect of Bombyx mori by Dimethyl Sulfoxide

    PubMed Central

    Yamamoto, Takayuki; Mase, Keisuke; Sawada, Hiroshi

    2013-01-01

    HCl treatment has been, for about 80 years, the primary method for the prevention of entry into embryonic diapauses of Bombyx mori. This is because no method is as effective as the HCl treatment. In this study, we discovered that dimethyl sulfoxide (DMSO) prevented entry into the diapause of the silkworm, Bombyx mori. The effect of diapause prevention was 78% as a result of treatment with 100% DMSO concentration, and the effect was comparable to that of the HCl treatment. In contrast, in the case of non-diapause eggs, hatchability was decreased by DMSO in a concentration-dependent manner. The effect of DMSO was restricted within 24 hours after oviposition of diapause eggs, and the critical period was slightly shorter than the effective period of the HCl treatment. DMSO analogs, such as dimethyl formamide (DMF) and dimethyl sulfide (DMS), did little preventive effect against the diapause. Furthermore, we also investigated the permeation effects of chemical compounds by DMSO. When treated with an inhibitor of protein kinase CK2 (CK2) dissolved in DMSO, the prevention rate of the diapause was less than 40%. This means that the inhibition effect by the CK2 inhibitor was the inhibition of embryonic development after diapause prevention by DMSO. These data suggest that DMSO has the effects of preventing from entering into the diapause and permeation of chemicals into diapause eggs. PMID:23675522

  8. Cytotoxic compounds against cancer cells from Bombyx mori inoculated with Cordyceps militaris.

    PubMed

    Qiu, Weitao; Wu, Jing; Choi, Jae-Hoon; Hirai, Hirofumi; Nishida, Hiroshi; Kawagishi, Hirokazu

    2017-06-01

    Two compounds, 3'-deoxyinosine and cordycepin, were isolated from Bombyx mori inoculated with Cordyceps militaris. In the bioassay examining cytotoxicity against cancer cells, both compounds showed toxicity against A549, PANC-1, and MCF-7 cancer cells.

  9. Investigation of Natural Bombyx mori Silk Fibroin Proteins Using INS

    NASA Astrophysics Data System (ADS)

    Crain, Christopher; Strange, Nicholas; Larese, J. Z.

    The mechanical properties of many protein comprised biomaterials are a direct reflection of non-covalent (i.e. weak) interacting ions such as F-actin in muscles, tubulin in the cytoskeleton of cells, viral capsids, and silk. Porter and Vollrath underscored the two main factors that are critical for understanding the high mechanical strength of silks: the nanoscale semi-crystalline folding structure, which gives it exceptional toughness and strength, and the degree of hydration of the disordered fraction, which acts to modify these properties. Understanding and controlling these two principal factors are the key to the functionality of protein elastomers, and render silk an ideal model protein for (bio)material design. We will describe our investigation of electrospun silk of the Bombyx mori (silk worm), using Inelastic Neutron Scattering (INS). These techniques were used to investigate the microscopic dynamics of the dry and hydrated protein.

  10. Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori).

    PubMed

    Tada, Minoru; Tatematsu, Ken-ichiro; Ishii-Watabe, Akiko; Harazono, Akira; Takakura, Daisuke; Hashii, Noritaka; Sezutsu, Hideki; Kawasaki, Nana

    2015-01-01

    In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO- and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity.

  11. Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

    PubMed Central

    Tada, Minoru; Tatematsu, Ken-Ichiro; Ishii-Watabe, Akiko; Harazono, Akira; Takakura, Daisuke; Hashii, Noritaka; Sezutsu, Hideki; Kawasaki, Nana

    2015-01-01

    In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO− and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity. PMID:26261057

  12. Recent progress in genome engineering techniques in the silkworm, Bombyx mori.

    PubMed

    Daimon, Takaaki; Kiuchi, Takashi; Takasu, Yoko

    2014-01-01

    Rapid advances in genome engineering tools, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced palindromic repeats/CRISPR-associated (CRISPR/Cas) system, have enabled efficient gene knockout experiments in a wide variety of organisms. Here, we review the recent progress in targeted gene disruption techniques in the silkworm, Bombyx mori. Although efficiency of targeted mutagenesis was very low in an early experiment using ZFNs, recent studies have shown that TALENs can induce highly efficient mutagenesis of desired target genes in Bombyx. Notably, mutation frequencies induced by TALENs can reach more than 50% of G0 gametes. Thus, TALENs can now be used as a standard tool for gene targeting studies, even when mutant phenotypes are unknown. We also propose guidelines for experimental design and strategy for knockout experiments in Bombyx. Genome editing technologies will greatly increase the usefulness of Bombyx as a model for lepidopteran insects, the major agricultural pests, and lead to sophisticated breeding of Bombyx for use in sericulture and biotechnology.

  13. Biotransformation effect of Bombyx Mori L. may play an important role in treating diabetic nephropathy.

    PubMed

    Zhang, Lei; Zhang, La; Li, Yin; Guo, Xin-Feng; Liu, Xu-Sheng

    2016-11-01

    Compared with herbal drugs, medicine processed from animals (animal medicine) was thought to have more bioactive substances and higher activities. Biotransformation effect often plays an important role in their effect. However, researches about effect of animal medicine on diabetic nephropathy and applying animal medicine as natural bio-transformer were seldom reported. The purpose of this paper was to reveal the use of Bombyx Mori L. on diabetic nephropathy from ancient to modern times. The classical literature indicated that Saosi Decoction (), which contains Bombyx Mori L. or silkworm cocoon, was applied to treat disorders congruent with modern disease diabetic nephropathy from the Ming to Qing Dynasty in ancient China. Modern studies showed that Bombyx Mori L. contains four main active constituents. Among these, 1-deoxynojirimycin (1-DNJ) and quercetin showed promising potential to be new agents in diabetic nephropathy treatment. The concentrations of 1-DNJ and the activities of quercetin in Bombyx Mori L. are higher than in mulberry leaves, because of the biotransformation in the Bombyx Mori L. body. However, these specifific components need further human and mechanistic studies to determine their therapeutic potential for this challenging condition.

  14. Retrotransposon "Qian" mediated segmental duplication in silkworm, Bombyx mori.

    PubMed

    Xu, Yunmin; Jiang, Ning; Zou, Ziliang; Tu, Zhijian; Chen, Anli; Zhao, Qiaoling; Xiang, Zhonghuai; He, Ningjia

    2014-03-01

    Transposable elements constitute a large fraction of the eukaryotic genomes. They have the potential to alter genome structure and play a major role in genome evolution. Here, we report a segmental duplication mediated by a novel long terminal repeat (LTR) retrotransposon as the cause of an egg-shell recessive lethal mutant (l-em mutant) in silkworm (Bombyx mori). The segmental duplication resulted in the duplication of six genes and the disruption of two genes. Disruption of BmEP80 (B. mori egg protein 80), a gene encoding a major egg-shell structure protein, is likely responsible for the lethal water-loss phenotype in the l-em/l-em mutant. Our data revealed that BmEP80 is present in the inner egg-shell layer and plays important roles in resistance to water efflux form eggs. A novel LTR retrotransposon (named as "Qian") was identified and the model for the Qian-mediated chromosomal segmental duplication was proposed. Detail biochemical and genomic analyses on the l-em mutant offer an opportunity to demonstrate that an LTR retrotransposon could trigger duplication of a chromosomal segment (∼96.3 kb) and confer novel phenotype.

  15. Characterization of Argonaute family members in the silkworm, Bombyx mori.

    PubMed

    Wang, Gen-Hong; Jiang, Liang; Zhu, Li; Cheng, Ting-Cai; Niu, Wei-Huan; Yan, Ya-Fei; Xia, Qing-You

    2013-02-01

    The Argonaute protein family is a highly conserved group of proteins, which have been implicated in RNA silencing in both plants and animals. Here, four members of the Argonaute family were systemically identified based on the genome sequence of Bombyx mori. Based on their sequence similarity, BmAgo1 and BmAgo2 belong to the Ago subfamily, while BmAgo3 and BmPiwi are in the Piwi subfamily. Phylogenetic analysis reveals that silkworm Argonaute family members are conserved in insects. Conserved amino acid residues involved in recognition of the 5' end of the small RNA guide strand and of the conserved (aspartate, aspartate and histidine [DDH]) motif present in their PIWI domains suggest that these four Argonaute family members may have conserved slicer activities. The results of microarray expression analysis show that there is a low expression level for B. mori Argonaute family members in different tissues and different developmental stages, except for BmPiwi. All four B. mori Argonaute family members are upregulated upon infection with B. mori nucleopolyhedrovirus. The complete coding sequence of BmPiwi, the homolog of Drosophila piwi, was cloned and its expression occurred mainly in the area where spermatogonia and spermatocytes appear. Our results provide an overview of the B. mori Argonaute family members and suggest that they may have multiple roles. In addition, this is also the first report, to our knowledge, of the response of RNA silencing machinery to DNA virus infection in insects.

  16. Aligning the proteome and genome of the silkworm, Bombyx mori.

    PubMed

    Zhang, Yaozhou; Xia, Qingyou; Xu, Jie; Chen, Jian; Nie, Zuoming; Wang, Dan; Zhang, Wenping; Chen, Jianqing; Zheng, Qingliang; Chen, Qing; Kong, Lingying; Ren, Xiaoyuan; Wang, Jiang; Lv, Zhengbing; Yu, Wei; Jiang, Caiying; Liu, Lili; Sheng, Qing; Jin, Yongfeng; Wu, Xiangfu

    2009-11-01

    A technology of mass spectrometry (MS) was used in this study for the large-scale proteomic identification and verification of protein-encoding genes present in the silkworm (Bombyx mori) genome. Peptide sequences identified by MS were compared with those from an open reading frame (ORF) library of the B. mori genome and a cDNA library, to validate the coding attributes of ORFs. Two databases were created. The first was based on a 9x draft sequence of the silkworm genome and contained 14,632 putative proteins. The second was based on a B. mori pupal cDNA library containing 3,187 putative proteins of at least 30 amino acid residues in length. A total of 81,000 peptide sequences with a threshold score of 60% were generated by the MS/MS analysis, and 55,400 of these were chosen for a sequence alignment. By searching these two databases, 6,649 and 250 proteins were matched, which accounted for approximately 45.4% and 7.8% of the peptide sequences and putative proteins, respectively. Further analyses carried out by several bioinformatic tools suggested that the matches included proteins with predicted transmembrane domains (1,393) and preproteins with a signal peptide (976). These results provide a fundamental understanding of the expression and function of silkworm proteins.

  17. Molecular Characterization of Endoplasmic Reticulum Oxidoreductin 1 from Bombyx mori.

    PubMed

    Seo, Minchul; Ryou, Hee-Joo; Yun, Eun-Young; Goo, Tae-Won

    2015-11-05

    We isolated a complementary DNA (cDNA) clone encoding endoplasmic reticulum oxidoreductin 1 (bERO1, a specific oxidant of protein disulfide isomerase (PDI)) from Bombyx mori. This protein has a putative open reading frame (ORF) of 489 amino acids and a predicted size of 57.4 kDa. Although bERO1 protein shares less than 57% amino acid sequence homology with other reported ERO1s, it contains two conserved redox active motifs, a Cys-X-X-X-X-Cys motif of N-terminal and Cys-X-X-Cys-X-X-Cys motif of C-terminal. Both motifs are typically present in ERO1 protein family members. The bEro1 mRNA expression was highest in posterior silk gland on the sixth day of the 5th instar larvae. Expression of bEro1 mRNA also markedly increased during endoplasmic reticulum (ER) stress induced by stimulation with antimycin, calcium ionophore A23187, dithiothreitol, H₂O₂, monencin, and tunicamycin. In addition, expression levels of bEro1 exactly coincided with that of bPdi. This is the first result suggesting that bERO1 plays an essential role in ER quality control through the combined activities of bERO1 and bPDI as a catalyst of protein folding in the ER and sustaining cellular redox homeostasis.

  18. A characterization of structural proteins expressed by Bombyx mori bidensovirus.

    PubMed

    Lü, Peng; Xing, Yali; Hu, Zhaoyang; Yang, Yanhua; Pan, Ye; Chen, Kangmin; Zhu, Feifei; Zhou, Yajing; Chen, Keping; Yao, Qin

    2017-03-01

    Bombyx mori bidensiovirus (BmBDV) is a species of Bidensovirus that has been was placed into a new genus within the new family Bidnaviridae by the International Committee on Taxonomy of Viruses. BmBDV causes fatal flacherie disease in silkworms, which causes large losses to the sericulture industry. BmBDV contains two sets of complementary linear single-stranded DNAs of approximately 6.5kb (viral DNA 1, VD1) and 6.0kb (viral DNA 2, VD2). VD1 and VD2 are encapsidated in separate icosahedral non-enveloped capsids, which are similar in size and shape. However, the strategies used to express BmBDV structural proteins remains unclear. In this work, a total of six structural proteins were separated by two-dimensional electrophoresis and shown to be encoded by the BmBDV VP gene via mass spectrometry. The transmission electron microscopy results showed that co-expression of the BmBDV VP and SP structural proteins in Spodoptera frugiperda sf9 cells resulted in the formation of 22-24nm virus-like particles. Furthermore, a mutation of the major structural protein-encoding VP gene, in which the second in-frame ATG codon was mutated to GCG, abrogated the production of several structural proteins, indicating that this strategy of expressing BmBDV VP is dependent on a leaky scanning translation mechanism.

  19. Structure refinement and diffuse streak scattering of silk (Bombyx mori).

    PubMed

    Takahashi, Y; Gehoh, M; Yuzuriha, K

    1999-01-01

    Reexamination of the crystal structure of silk (Bombyx mori) was carried out by X-ray diffraction method. Four molecular chains are contained in the rectangular unit cell with parameters, a = 9.38 A, b = 9.49 A, and c (fiber axis) = 6.98 A, and the space group P2(1)-C(2)2. Silk assumes the statistical crystal structure, in which two antipolar-antiparallel sheet structures with different orientations statistically occupy a crystal site with the ratio 2:1. The molecular conformation is essentially the same pleated sheet structure as Marsh, Corey and Pauling proposed. However, the sheet structure formed by hydrogen bonds assumes the antipolar antiparallel structure different from that proposed by Marsh, Corey and Pauling, in which the methyl groups of alanine residues alternately point to both sides of the sheet structure along the hydrogen bonding direction. The crystalline region of silk is composed of stacking of two antipolar antiparallel sheet structures with different orientations.

  20. Ultrathin Free-Standing Bombyx mori Silk Nanofibril Membranes.

    PubMed

    Ling, Shengjie; Jin, Kai; Kaplan, David L; Buehler, Markus J

    2016-06-08

    We report a new ultrathin filtration membrane prepared from silk nanofibrils (SNFs), directly exfoliated from natural Bombyx mori silk fibers to retain structure and physical properties. These membranes can be prepared with a thickness down to 40 nm with a narrow distribution of pore sizes ranging from 8 to 12 nm. Typically, 40 nm thick membranes prepared from SNFs have pure water fluxes of 13 000 L h(-1) m(-2) bar(-1), more than 1000 times higher than most commercial ultrathin filtration membranes and comparable with the highest water flux reported previously. The commercial membranes are commonly prepared from polysulfone, poly(ether sulfone), and polyamide. The SNF-based ultrathin membranes exhibit efficient separation for dyes, proteins, and colloids of nanoparticles with at least a 64% rejection of Rhodamine B. This broad-spectrum filtration membrane would have potential utility in applications such as wastewater treatment, nanotechnology, food industry, and life sciences in part due to the protein-based membrane polymer (silk), combined with the robust mechanical and separation performance features.

  1. Accumulation of 1-deoxynojirimycin in silkworm, Bombyx mori L.

    PubMed Central

    Yin, Hao; Shi, Xin-qin; Sun, Bo; Ye, Jing-jing; Duan, Zu-an; Zhou, Xiao-ling; Cui, Wei-zheng; Wu, Xiao-feng

    2010-01-01

    1-deoxynojirimycin (1-DNJ) contents in the silkworm, Bombyx mori, at different developmental stages and tissues were investigated by using reverse-phase high-performance liquid chromatography. The 1-DNJ contents of silkworm larvae change significantly with their developmental stages. The male larvae showed higher accumulation efficiency of 1-DNJ than the females and also a significant variation was observed among the silkworm strains. The present results show that tissue distribution of 1-DNJ was significantly higher in blood, digestive juice, and alimentary canal, but no 1-DNJ was observed in the silkgland. Moreover, 1-DNJ was not found in silkworms fed with artificial diet that does not contain mulberry leaf powder. This proves that silkworms obtain 1-DNJ from mulberry leaves; they could not synthesize 1-DNJ by themselves. The accumulation and excretion of 1-DNJ change periodically during the larval stage. There was no 1-DNJ in the newly-hatched larvae and 1-DNJ was mainly accumulated during the early and middle stages of every instar, while excreted at later stages of larval development. Further, it is possible to extract 1-DNJ from the larval feces and it is optimal to develop the 1-DNJ related products for diabetic auxiliary therapy. PMID:20349525

  2. Mechanism of fluorescent cocoon sex identification for silkworms Bombyx mori.

    PubMed

    Zhang, YuQing; Yu, XiaoHua; Shen, WeiDe; Ma, YongLei; Zhou, LiXia; Xu, NaiXi; Yi, ShuQian

    2010-11-01

    By using silkworms, Bombyx mori, fluorescent cocoon sex identification (FCSI) as an experimental material, direct fluorescence spectrometry of the cocoon surface indicates that the fluorescent color of silkworm cocoons is made up of two peaks of yellow and blue-purple fluorescence emission. The fluorescent difference between male and female cocoons is attributed to the differential absorption of yellow fluorescent substances by the midgut tissue of 5th instar female silkworms. Thin layer chromatography (TLC) and fluorescent spectra indicate that blue-purple fluorescent substances are composed of at least five blue-purple fluorescent pigments, and yellow fluorescent substances are made up of at least three. UV spectra and AlCl₃ color reaction show that the three fluorescent yellow pigments are flavonoids or their glycosides. Silkworm FCSI is due to selective absorption or accumulation of the yellow fluorescent pigments by the posterior midgut cells of female 5th instar larvae. The cells of the FCSI silkworm midgut, especially the cylinder intestinal cells of the posterior midgut have a component which is a yellow fluorescent pigment-specific binding protein that may be vigorously expressed in the 5th instar larvae.

  3. Genome engineering and parthenocloning in the silkworm, Bombyx mori.

    PubMed

    Zabelina, Valeriya; Klymenko, Vyacheslav; Tamura, Toshiki; Doroshenko, Karina; Liang, Haoyuan; Sezutsu, Hideki; Sehnal, František

    2015-09-01

    Genetic engineering of the silkworm, Bombyx mori, opens door to the production of new kinds of silk and to the use of silkworms as proteosynthetic bioreactors. The insertion of foreign genes into silkworm genome and the control of their expression by diverse promoters have become possible but are not yet efficient enough for commercial use. Several methods of gene targeting are being developed to minimize position effect on transgene expression and facilitate cloning. Parthenocloning can be exploited to conserve genetic traits and improve selection and amplification of clones containing genes of interest. Some silkworm clones have been bred for decades as genetically stable female stocks whose unfertilized eggs are induced to develop by heat-shock treatment. Any exclusively female generation contains exact copies of the maternal clone-founder genome. Ovaries transplanted in either direction between the standard and the parthenogenetic genotypes yield eggs capable of parthenocloning. In addition, use ofmale larvae as ovary recipients eliminates diapause in eggs produced in the implants. Unfertilized eggs of some silkworm clones respond also to the cold-shock treatment by producing homozygous fertile sons; cloned females can be crossed with their parthenogenetic sons to obtain progeny homozygous for the transgene in both sexes. Rational exploitation of available parthenozygous pools and the use of parthenocloning methods enable rapid fixation and maintenance of the desired genotypes.

  4. Molecular Characterization of Endoplasmic Reticulum Oxidoreductin 1 from Bombyx mori

    PubMed Central

    Seo, Minchul; Ryou, Hee-Joo; Yun, Eun-Young; Goo, Tae-Won

    2015-01-01

    We isolated a complementary DNA (cDNA) clone encoding endoplasmic reticulum oxidoreductin 1 (bERO1, a specific oxidant of protein disulfide isomerase (PDI)) from Bombyx mori. This protein has a putative open reading frame (ORF) of 489 amino acids and a predicted size of 57.4 kDa. Although bERO1 protein shares less than 57% amino acid sequence homology with other reported ERO1s, it contains two conserved redox active motifs, a Cys-X-X-X-X-Cys motif of N-terminal and Cys-X-X-Cys-X-X-Cys motif of C-terminal. Both motifs are typically present in ERO1 protein family members. The bEro1 mRNA expression was highest in posterior silk gland on the sixth day of the 5th instar larvae. Expression of bEro1 mRNA also markedly increased during endoplasmic reticulum (ER) stress induced by stimulation with antimycin, calcium ionophore A23187, dithiothreitol, H2O2, monencin, and tunicamycin. In addition, expression levels of bEro1 exactly coincided with that of bPdi. This is the first result suggesting that bERO1 plays an essential role in ER quality control through the combined activities of bERO1 and bPDI as a catalyst of protein folding in the ER and sustaining cellular redox homeostasis. PMID:26556347

  5. Molecular Evidence of RNA Editing in Bombyx Chemosensory Protein Family

    PubMed Central

    Xuan, Ning; Bu, Xun; Liu, Yan Yan; Yang, Xue; Liu, Guo Xia; Fan, Zhong Xue; Bi, Yu Ping; Yang, Lian Qun; Lou, Qi Nian; Rajashekar, Balaji; Leppik, Getter; Kasvandik, Sergo; Picimbon, Jean-François

    2014-01-01

    Chemosensory proteins (CSPs) are small scavenger proteins that are mainly known as transporters of pheromone/odor molecules at the periphery of sensory neurons in the insect antennae and in the producing cells from the moth female pheromone gland. Sequencing cDNAs of RNA encoding CSPs in the antennae, legs, head, pheromone gland and wings from five single individual adult females of the silkworm moth Bombyx mori showed that they differed from genomic sequences by subtle nucleotide replacement (RDD). Both intronless and intronic CSP genes expressed RDDs, although in different rates. Most interestingly, in our study the degree of RDDs in CSP genes were found to be tissue-specific. The proportion of CSP-RDDs was found to be significantly much higher in the pheromone gland. In addition, Western blot analysis of proteins in different tissues showed existence of multiple CSP protein variant chains particularly found in the pheromone gland. Peptide sequencing demonstrated the occurrence of a pleiad of protein variants for most of all BmorCSPs from the pheromone gland. Our findings show that RNA editing is an important feature in the expression of CSPs and that a high variety of RDDs is found to expand drastically thus altering the repertoire of CSP proteins in a tissue-specific manner. PMID:24551045

  6. Accumulation of 1-deoxynojirimycin in silkworm, Bombyx mori L.

    PubMed

    Yin, Hao; Shi, Xin-qin; Sun, Bo; Ye, Jing-jing; Duan, Zu-an; Zhou, Xiao-ling; Cui, Wei-zheng; Wu, Xiao-feng

    2010-04-01

    1-deoxynojirimycin (1-DNJ) contents in the silkworm, Bombyx mori, at different developmental stages and tissues were investigated by using reverse-phase high-performance liquid chromatography. The 1-DNJ contents of silkworm larvae change significantly with their developmental stages. The male larvae showed higher accumulation efficiency of 1-DNJ than the females and also a significant variation was observed among the silkworm strains. The present results show that tissue distribution of 1-DNJ was significantly higher in blood, digestive juice, and alimentary canal, but no 1-DNJ was observed in the silkgland. Moreover, 1-DNJ was not found in silkworms fed with artificial diet that does not contain mulberry leaf powder. This proves that silkworms obtain 1-DNJ from mulberry leaves; they could not synthesize 1-DNJ by themselves. The accumulation and excretion of 1-DNJ change periodically during the larval stage. There was no 1-DNJ in the newly-hatched larvae and 1-DNJ was mainly accumulated during the early and middle stages of every instar, while excreted at later stages of larval development. Further, it is possible to extract 1-DNJ from the larval feces and it is optimal to develop the 1-DNJ related products for diabetic auxiliary therapy.

  7. Two Tor genes in the silkworm Bombyx mori.

    PubMed

    Zhou, S; Zhou, Q; Liu, Y; Wang, S; Wen, D; He, Q; Wang, W; Bendena, W G; Li, S

    2010-12-01

    Target of rapamycin (TOR), a member of the phosphatidylinositol kinase-related kinase family, plays a critical role in the regulation of growth, metabolism, development and survival, at both the cellular and the organismal levels. Two paralogous Tor genes, BmTor1 and BmTor2, were identified as a pair of inverted repeats in the genome of the silkworm Bombyx mori. The synteny of BmTor1 and CG8360 indicates that BmTor1 is the orthologue while BmTor2 is a duplicate. Analyses of the two BmTor genes at both the nucleotide and amino acid levels reveal that they are evolutionally and structurally conserved. The two BmTor genes had similar expression patterns of tissue distribution with highest levels in the nervous system, and nearly identical developmental change profiles with maximal levels during the 4(th) -larval-moulting and the larval-pupal transition stages. Furthermore, both BmTor genes were up-regulated by either starvation or the moulting hormone 20-hydroxyecdysone (20E), while BmTor2 was more sensitive to both treatments than BmTor1. For the first time, we have identified two copies of the Tor gene in a higher eukaryote, which are induced by starvation and 20E during the larval moulting and the larval-pupal transition stage. © 2010 The Authors. Journal compilation © 2010 The Royal Entomological Society.

  8. Metabonomic analysis of Bombyx mori (Heterocera: Bombysidae) treated with acetaminophen.

    PubMed

    Yin, W M; Xu, X; He, Y; Wei, G B; Sima, Y H; Shi-Qing, Xu

    2014-01-01

    The feasibility of using Bombyx mori as model animal is attracting more attention. Whether the effect of drugs on the metabolite profiling was consistent with those in mammals was an aspect to evaluate the feasibility of B. mori as model animal. In this study, we used acetaminophen to treat Dazao fifth-instar B. mori, and its metabolites in hemolymph were detected by gas chromatography-mass spectrometry. The corresponding data were processed and analyzed by total model analysis, principal component analysis, partial least squares-discriminant analysis, orthogonal partial least squares-discriminant analysis, and finally, the difference metabolites between acetaminophen group and control group were selected and identified by our reference material database and the National Institute of Standard and Technology database. The results showed that acetaminophen administration induced elevation of metabolites related to energy source, the intermediate of cholesterol synthesis, and the metabolites related to melanization and also induced the decrease of metabolites in pathway of Krebs cycle, the cholesterol, and sitosterol, which suggested that acetaminophen administration inhibited energy metabolism and promoted the expenditure and imbalance of hormone and melanization.

  9. Identification and expression analysis of ras gene in silkworm, Bombyx mori.

    PubMed

    Ogura, Takehiko; Tan, Anjiang; Tsubota, Takuya; Nakakura, Takayo; Shiotsuki, Takahiro

    2009-11-25

    Ras proteins play important roles in development especially for cell proliferation and differentiation in various organisms. However, their functions in the most insect species are still not clear. We identified three ras cDNAs from the silk worm, Bombyx mori. These sequences corresponded to three Ras of Drosophila melanogaster, but not to three mammalian Ras (H-Ras, K-Ras, N-Ras). Subsequently, the expression profiles of ras were investigated by quantitative real-time PCR using whole body of individuals from the embryonic to adult stages, and various tissues of 4th and 5th instar larvae. Each of three Bombyx ras showed different expression patterns. We also showed membrane localization of their products. These results indicate that the three Bombyx Ras are functional and have different roles.

  10. Pigmentary analysis of eggs of the silkworm Bombyx mori.

    PubMed

    Zhang, Haiyan; Lin, Ying; Shen, Guanwang; Tan, Xue; Lei, Chao; Long, Wei; Liu, Hongling; Zhang, Yandi; Xu, Yinying; Wu, Jinxin; Gu, Jianjian; Xia, Qingyou; Zhao, Ping

    2017-08-01

    Ommochromes are major pigments involved in coloration of eggs, eyes, wings, and epidermis of insects. Bombyx mori (silkworm) eggs contain a mixture of ommochrome pigments and their precursors. Here, we analyzed the pigment composition of every egg color strain using egg color mutants (w-2, pe, and re) and wild-type strains (dazao and C108) by using full wavelength scanning and high-performance liquid chromatography. We identified ommochrome pigments and their precursors in pigment extracts from non-diapause eggs and diapause eggs, and found that the quantities of ommochrome precursor 3-hydroxy-kynurenine were much higher in the diapause eggs. Ommochrome pigments were absent in the non-diapause eggs. We analyzed the pigment composition of every egg color strain and found an accumulation of 3-hydroxy-kynurenine and absence of ommochromes in the yellow eggs (w-2 and pe), suggesting that the essential factors for ommochrome biosynthesis are high levels of 3-hydroxy-kynurenine, enzymes for ommochrome synthesis and transferase, and spermatiation. Moreover, we confirmed that both decarboxylated xanthommatin and xanthommatin are major ommochrome pigments, and the quantity of decarboxylated xanthommatin is much higher than that of xanthommatin in silkworm eggs. Since ommochrome pigments can change color under oxidative/reductive conditions and the egg color mutant re turns crimson when preserved at a low temperature for a few weeks, we used an oxidation-reduction reaction in vitro to explore mechanisms behind the pigment-based color change. Specifically, during diapause, the contents of decarboxylated xanthommatin and xanthommatin are increased, and the ommochrome pigments convert into their reduced forms. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Identification of circular RNA in the Bombyx mori silk gland.

    PubMed

    Gan, Huaiyan; Feng, Tieshan; Wu, Yuqian; Liu, Chun; Xia, Qingyou; Cheng, Tingcai

    2017-10-01

    Bombyx mori is an economically important holometabolous lepidopteran insect. In B. mori endogenous noncoding RNAs such as microRNAs (miRNAs) and Piwi-interacting RNAs play crucial biological functions in metamorphosis and sex determination. In addition, circular RNAs (circRNAs) have been recently identified as noncoding RNAs in most common model organisms and show potential as gene regulators. However, to date, there have been few studies on the circRNAs present in the B. mori genome conducted to date. Here, we identified 3916 circRNAs by deep circular transcriptome sequencing using the silk gland of B. mori. 3155 circRNAs were found to be derived from 1727 parental genes. The circRNAs displayed tissue-specific expression between the middle silk gland (MSG) and posterior silk gland (PSG), with 2532 and 880 being upregulated circRNAs in the MSG and PSG, respectively. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that the parental genes from the MSG and PSG were generally annotated to similar categories and pathways. The interaction network of circRNAs and miRNAs showed that circRNAs might act as miRNA sponges or interact with miRNAs in some other way. Overall, the results revealed the complicated patterns of circRNAs in the B. mori silk gland providing a new angle from which to explore the mechanisms of complex gene regulation and efficient silk protein synthesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. The dynamic landscape of gene regulation during Bombyx mori oogenesis.

    PubMed

    Zhang, Qiang; Sun, Wei; Sun, Bang-Yong; Xiao, Yang; Zhang, Ze

    2017-09-11

    Oogenesis in the domestic silkworm (Bombyx mori) is a complex process involving previtellogenesis, vitellogenesis and choriogenesis. During this process, follicles show drastic morphological and physiological changes. However, the genome-wide regulatory profiles of gene expression during oogenesis remain to be determined. In this study, we obtained time-series transcriptome data and used these data to reveal the dynamic landscape of gene regulation during oogenesis. A total of 1932 genes were identified to be differentially expressed among different stages, most of which occurred during the transition from late vitellogenesis to early choriogenesis. Using weighted gene co-expression network analysis, we identified six stage-specific gene modules that correspond to multiple regulatory pathways. Strikingly, the biosynthesis pathway of the molting hormone 20-hydroxyecdysone (20E) was enriched in one of the modules. Further analysis showed that the ecdysteroid 20-hydroxylase gene (CYP314A1) of steroidgenesis genes was mainly expressed in previtellogenesis and early vitellogenesis. However, the 20E-inactivated genes, particularly the ecdysteroid 26-hydroxylase encoding gene (Cyp18a1), were highly expressed in late vitellogenesis. These distinct expression patterns between 20E synthesis and catabolism-related genes might ensure the rapid decline of the hormone titer at the transition point from vitellogenesis to choriogenesis. In addition, we compared landscapes of gene regulation between silkworm (Lepidoptera) and fruit fly (Diptera) oogeneses. Our results show that there is some consensus in the modules of gene co-expression during oogenesis in these insects. The data presented in this study provide new insights into the regulatory mechanisms underlying oogenesis in insects with polytrophic meroistic ovaries. The results also provide clues for further investigating the roles of epigenetic reconfiguration and circadian rhythm in insect oogenesis.

  13. Anterior and posterior centers jointly regulate Bombyx embryo body segmentation.

    PubMed

    Nakao, Hajime

    2012-11-15

    Insect embryo segmentation is largely divided into long and short germ types. In the long germ type, each segment primordium is represented on a large embryonic rudiment of the blastoderm, and segmental patterning occurs nearly simultaneously in the syncytium. In the short germ type, however, only anterior segments are represented in the small embryonic rudiment, usually located on the egg posterior, and the rest of the segments are added sequentially from the posterior growth zone in a cellular context. The long germ type is thought to have evolved from the short germ type. It is proposed that this transition, which appears to have occurred multiple times over the course of evolution, was realized through the acquisition of a localized anterior instruction center. Here, I examined the early segmentation process in the silkmoth Bombyx mori, a lepidopteran insect, in which the mechanisms of anterior-posterior (AP) axis formation have not been well analyzed. In this insect, both the long germ and short germ features have been reported. The mRNAs for two key genes involved in insect AP axis formation, orthodenticle (Bm-otd) and caudal (Bm-cad), are localized maternally in the germ anlage, where they act as anterior and posterior instruction centers, respectively. RNAi studies indicate that, while Bm-cad affects the formation of all the even skipped (Bm-eve) stripes, there is also anterior Bm-eve stripe formation activity that involves Bm-otd. Thus, there is redundancy in Bm-eve stripe formation activity that must be coordinated. Some genetic interactions, identified either experimentally or hypothetically, are also introduced, which might enable robust AP formation in this organism.

  14. Effect of fortification of Mulberry leaves with homeopathic drug Nux vomica on Bombyx mori. L.

    PubMed

    Hiware, C J

    2006-07-01

    Silk worm (Bombyx mori L.) larvae were fed on Mulberry leaves treated with Nux vomica mother tincture. The impact on larval, cocoon, shell and pupal weight, silk ratio, average filament length and denier, and number of breakages during reeling were investigated. The results were positive in all parameters under study except cocoon weight, pupal weight, and the average denier of the filament.

  15. Effect of crude extract of Bombyx mori coccoons in hyperlipidemia and atherosclerosis

    PubMed Central

    Ali, Mir Mahdi; Arumugam, Sarasa Bharati A.

    2011-01-01

    The silkworm is the larva or caterpillar of the domesticated silkmoth, Bombyx mori and being a primary producer of silk is an economically important insect. These days the silk is emerging as a resource for solving a broad range of biological problems. The silk (Abresham) is popularly known as Abresham muqriz (muqriz means cut) in Unani medicine. Its cocoons are extensively used as an ingredient of various Unani formulations like Khameer-E- Abresham Sada, Khameere Abresham Hakeem Arshad Wala, Khameere Abresham Ood Mastagi Wala etc. and are used to treat many cardiac and nervous disorders. The hypolipidemic activity of this drug, along with Nepata Hindostana (Badranjboya) and Terminalia Arjuna (Arjan) has been documented. But action of extract of Bombyx mori cocoons as a single drug is not documented. That's why; it was decided to study its effect on hyperlipidemia and atherosclerosis. The Male New Zealand White rabbits all of 1.5kgs were selected for the study. After stabilization period (2 weeks) the rabbits were divided into 3 groups (Group I - Control, Group II Lesion Control and Group III treated with extract of Bombyx mori silk cocoon). Hyperlipidemia and atherosclerosis were induced with 1% cholesterol diet. After induction of hyperlipidemia and atherosclerosis for twelve weeks, Group III rabbits were treated with Bombyx mori for 6 weeks (45 days). A significant decrease in hyperlipidemia was seen within 4 weeks of treatment. Histopathologically, the atherosclerotic plaques showed reduction in size. The third group showed a significant increase in the body weight and also an increase in the HDL cholesterol levels. The study concludes that extract of Bombyx mori cocoons has a significant effect on hypercholesterolemia and atherosclerosis probably because of its antioxidant and hypolipidemic effect. PMID:21760692

  16. Effect of crude extract of Bombyx mori coccoons in hyperlipidemia and atherosclerosis.

    PubMed

    Ali, Mir Mahdi; Arumugam, Sarasa Bharati A

    2011-04-01

    The silkworm is the larva or caterpillar of the domesticated silkmoth, Bombyx mori and being a primary producer of silk is an economically important insect. These days the silk is emerging as a resource for solving a broad range of biological problems. The silk (Abresham) is popularly known as Abresham muqriz (muqriz means cut) in Unani medicine. Its cocoons are extensively used as an ingredient of various Unani formulations like Khameer-E- Abresham Sada, Khameere Abresham Hakeem Arshad Wala, Khameere Abresham Ood Mastagi Wala etc. and are used to treat many cardiac and nervous disorders. The hypolipidemic activity of this drug, along with Nepata Hindostana (Badranjboya) and Terminalia Arjuna (Arjan) has been documented. But action of extract of Bombyx mori cocoons as a single drug is not documented. That's why; it was decided to study its effect on hyperlipidemia and atherosclerosis. The Male New Zealand White rabbits all of 1.5kgs were selected for the study. After stabilization period (2 weeks) the rabbits were divided into 3 groups (Group I - Control, Group II Lesion Control and Group III treated with extract of Bombyx mori silk cocoon). Hyperlipidemia and atherosclerosis were induced with 1% cholesterol diet. After induction of hyperlipidemia and atherosclerosis for twelve weeks, Group III rabbits were treated with Bombyx mori for 6 weeks (45 days). A significant decrease in hyperlipidemia was seen within 4 weeks of treatment. Histopathologically, the atherosclerotic plaques showed reduction in size. The third group showed a significant increase in the body weight and also an increase in the HDL cholesterol levels. The study concludes that extract of Bombyx mori cocoons has a significant effect on hypercholesterolemia and atherosclerosis probably because of its antioxidant and hypolipidemic effect.

  17. The Origin and Dispersal of the Domesticated Chinese Oak Silkworm, Antheraea pernyi, in China: A Reconstruction Based on Ancient Texts

    PubMed Central

    Liu, Yanqun; Li, Yuping; Li, Xisheng; Qin, Li

    2010-01-01

    Sericulture is one of the great inventions of the ancient Chinese. Besides the mulberry silkworm (Bombyx mori), Chinese farmers developed rearing of the Chinese oak silkworm (Antheraea pernyi) about 400 years ago. In this paper, the historic records of the origins and dispersal of the domesticated Chinese oak silkworm in China are summarized. The first document with clearly recorded oak silkworm artificial rearing appeared in 1651, although Chinese oak silkworm was documented in about 270 AD. All of the evidence in the available historic records suggests that the domesticated Chinese oak silkworm originated in central and southern areas of Shandong Province in China around the 16th century, and then was introduced directly and indirectly by human commerce into the present habitations in China after the late 17th century. The results strongly support the hypothesis that only one geographically distinct event occurred in domestication of the modern Chinese oak silkworm. PMID:21062145

  18. The origin and dispersal of the domesticated Chinese oak silkworm, Antheraea pernyi, in China: a reconstruction based on ancient texts.

    PubMed

    Liu, Yanqun; Li, Yuping; Li, Xisheng; Qin, Li

    2010-01-01

    Sericulture is one of the great inventions of the ancient Chinese. Besides the mulberry silkworm (Bombyx mori), Chinese farmers developed rearing of the Chinese oak silkworm (Antheraea pernyi) about 400 years ago. In this paper, the historic records of the origins and dispersal of the domesticated Chinese oak silkworm in China are summarized. The first document with clearly recorded oak silkworm artificial rearing appeared in 1651, although Chinese oak silkworm was documented in about 270 AD. All of the evidence in the available historic records suggests that the domesticated Chinese oak silkworm originated in central and southern areas of Shandong Province in China around the 16th century, and then was introduced directly and indirectly by human commerce into the present habitations in China after the late 17th century. The results strongly support the hypothesis that only one geographically distinct event occurred in domestication of the modern Chinese oak silkworm.

  19. Thymosin From Bombyx mori Is Down-Regulated in Expression by BmNPV Exhibiting Antiviral Activity

    PubMed Central

    Zhang, Chen; Wang, Yongdi; Fang, Qiang; Xu, Minlin; Lv, Mengyuan; Liao, Jinxu; Li, Si; Nie, Zuoming; Zhang, Wenping

    2016-01-01

    Thymosins have been highly conserved during evolution. These hormones exist in many animal species and play an essential role in many biological events. However, little is known regarding the physiological function of silkworm Bombyx mori thymosin (BmTHY). In this study, we investigated the expression pattern of BmTHY in a Bombyx mori larval ovarian cell line (BmN) challenged with Bombyx mori nuclear polyhydrosis virus (BmNPV) and the antiviral effect of recombinant BmTHY (rBmTHY) for Bombyx mori against BmNPV. Western-blot assay and qRT-PCR analysis revealed that the level of BmTHY protein expression and transcription decreased over time when BmN cells were infected by BmNPV. Treatment with endotoxin-free rBmTHY led to a significant reduction in viral titer in the supernatant of BmN cells challenged with BmNPV. The results from antiviral tests performed in vitro and in vivo showed that endotoxin-free rBmTHY improved the survival rate of Bombyx mori infected with BmNPV. These findings suggest that BmTHY exerts immunomodulatory effects on Bombyx mori, rendering them resistant to viral infection. PMID:27432352

  20. Co-sensitization to silkworm moth (Bombyx mori) and 9 inhalant allergens among allergic patients in Guangzhou, Southern China.

    PubMed

    Sun, Baoqing; Zheng, Peiyan; Wei, Nili; Huang, Huimin; Zeng, Guangqiao

    2014-01-01

    This study aimed to investigate the profile of sensitization to silkworm moth (Bombyx mori) and other 9 common inhalant allergens among patients with allergic diseases in southern China. A total of 175 patients were tested for serum sIgE against silkworm moth in addition to combinations of other allergens: Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis, Blattella germanica, Periplaneta americana, cat dander, dog dander, Aspergillus fumigatus and Artemisia vulgaris by using the ImmunoCAP system. Correlation between sensitization to silkworm moth and to the other allergens was analyzed. Of the 175 serum samples tested, 86 (49.14%) were positive for silkworm moth sIgE. With high concordance rates, these silkworm moth sensitized patients were concomitantly sensitized to Dermatophagoides pteronyssinus (94.34%), Dermatophagoides farinae (86.57%), Blomia tropicalis (93.33%), Blattella germanica (96.08%), and Periplaneta americana (79.41%). Moreover, there was a correlation in serum sIgE level between silkworm moth and Dermatophagoides pteronyssinus (r = 0.518), Dermatophagoides farinae (r = 0.702), Blomia tropicalis (r = 0.701), Blattella germanica (r = 0.878), and Periplaneta americana (r = 0.531) among patients co-sensitized to silkworm moth and each of these five allergens. In southern Chinese patients with allergic diseases, we showed a high prevalence of sensitization to silkworm moth, and a co-sensitization between silkworm moth and other five common inhalant allergens. Further serum inhibition studies are warranted to verify whether cross-reactivity exists among these allergens.

  1. Injury-induced rapid activation of MAPK signaling in dechorionated eggs and larvae of the silkworm Bombyx mori.

    PubMed

    Gu, Shi-Hong; Chen, Chien-Hung

    2017-04-01

    Previous study showed that diapause in Bombyx mori eggs can be terminated by dechorionation and that activation in the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) in dechorionated cultured eggs is involved in diapause termination. In the present study, the possible mechanism underlying activation of ERK upon dechorionation was further investigated. Results showed that mechanical injury of diapause eggs without medium incubation also resulted in rapid increase in the phospho-ERK levels and that injury increased the phospho-ERK levels at different stages of both diapause eggs and eggs in which diapause initiation was prevented by HCl. Effects of anaerobiosis on dechorionation-stimulated phospho-ERK levels showed that the mechanical injury itself but not the dramatic increase in oxygen uptake upon injury is involved in a rapid activation of ERK. Chemical anaerobiosis on dechorionation-stimulated phospho-ERK levels and the in vivo effect of anaerobiosis showed that the supply of oxygen also plays a role in ERK signaling. In addition, injury induced the phosphorylation of c-jun N-terminal kinases (JNKs) and p38 kinase, components of two parallel MAPK pathways. A kinase assay showed a dramatic increase in JNK kinase activity in egg lysates upon injury. When newly hatched first instar larvae were injured, an increase in the phospho-ERK levels similar to that in dechorionated eggs was observed. From the results, we hypothesize that the injury-induced rapid activation of MAPK signaling, which serves as a natural signal for embryonic development, is related to diapause termination in dechorionated eggs. © 2015 Institute of Zoology, Chinese Academy of Sciences.

  2. Co-Sensitization to Silkworm Moth (Bombyx mori) and 9 Inhalant Allergens among Allergic Patients in Guangzhou, Southern China

    PubMed Central

    Wei, Nili; Huang, Huimin; Zeng, Guangqiao

    2014-01-01

    Objectives This study aimed to investigate the profile of sensitization to silkworm moth (Bombyx mori) and other 9 common inhalant allergens among patients with allergic diseases in southern China. Methods A total of 175 patients were tested for serum sIgE against silkworm moth in addition to combinations of other allergens: Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis, Blattella germanica, Periplaneta americana, cat dander, dog dander, Aspergillus fumigatus and Artemisia vulgaris by using the ImmunoCAP system. Correlation between sensitization to silkworm moth and to the other allergens was analyzed. Results Of the 175 serum samples tested, 86 (49.14%) were positive for silkworm moth sIgE. With high concordance rates, these silkworm moth sensitized patients were concomitantly sensitized to Dermatophagoides pteronyssinus (94.34%), Dermatophagoides farinae (86.57%), Blomia tropicalis (93.33%), Blattella germanica (96.08%), and Periplaneta americana (79.41%). Moreover, there was a correlation in serum sIgE level between silkworm moth and Dermatophagoides pteronyssinus (r = 0.518), Dermatophagoides farinae (r = 0.702), Blomia tropicalis (r = 0.701), Blattella germanica (r = 0.878), and Periplaneta americana (r = 0.531) among patients co-sensitized to silkworm moth and each of these five allergens. Conclusion In southern Chinese patients with allergic diseases, we showed a high prevalence of sensitization to silkworm moth, and a co-sensitization between silkworm moth and other five common inhalant allergens. Further serum inhibition studies are warranted to verify whether cross-reactivity exists among these allergens. PMID:24787549

  3. BmCalpains are involved in autophagy and apoptosis during metamorphosis and after starvation in Bombyx mori.

    PubMed

    Yi, Hui-Yu; Yang, Wan-Ying; Wu, Wen-Mei; Li, Xing-Xia; Deng, Xiao-Juan; Li, Qing-Rong; Cao, Yang; Zhong, Yang-Jin; Huang, Ya-Dong

    2016-11-07

    Apoptosis and autophagy play crucial roles during Bombyx mori metamorphosis and in response to various adverse conditions, including starvation. Recently, calpain, one of the major intracellular proteases, has been reported to be involved in apoptosis and autophagy in mammals. BmATG5 and BmATG6 have been identified to mediate apoptosis following autophagy induced by 20-hydroxyecdysone and starvation in B. mori. However, B. mori calpains and their functions remain unclear. In this study, phylogenetic analysis of calpains from B. mori, Drosophila melanogaster and Homo sapiens were performed and the results showed distinct close relationships of BmCalpain-A/B with DmCalpain-A/B, BmCalpain-C with DmCalpain-C, and BmCalpain-7 with HsCalpain-7. Then, the expression profiles of BmCalpains were analyzed by quantitative real-time polymerase chain reaction, and results showed that expression of BmCalpain-A/B, BmCalpain-C and BmCalpain-7 was significantly increased during B. mori metamorphosis and induced in the fat body and midgut of starved larvae, which is consistent with the expression profiles of BmAtg5, BmAtg6 and BmCaspase-1. Moreover, the apoptosis-associated cleavage of BmATG6 in Bm-12 cells was significantly enhanced when BmCalpain-A/B and BmCalpain-7 were induced by starvation, and was partially inhibited by the inhibitor of either calpain or caspase, but completely inhibited when both types of inhibitors were applied together. Our results indicated that BmCalpains, including BmCalpain-A/B, -C and -7, may be involved in autophagy and apoptosis during B. mori metamorphosis and after starvation, and may also contribute to the apoptosis-associated cleavage of BmATG6. © 2016 Institute of Zoology, Chinese Academy of Sciences.

  4. Silvernanotherapy on the viral borne disease of silkworm Bombyx mori L.

    NASA Astrophysics Data System (ADS)

    Govindaraju, K.; Tamilselvan, S.; Kiruthiga, V.; Singaravelu, G.

    2011-12-01

    Antiviral assays of chemically and biologically synthesized silver nanoparticles were made against BmNPV ( Bombyx mori Nuclear Polyhedrosis Virus). Reduction of silver ions by sodium citrate and Spirulina platensis led to the formation of spherical silver nanoparticles of 40-60 and 7-16 nm size. Single cell protein ( Spirulina platensis)-synthesized silver nanoparticles showed the strongest antiviral activity. Immunological studies made on the silkworm Bombyx mori disclosed that a significant increase in the total hemocyte count and differential hemocyte count due to S. platensis-synthesized silver nanoparticles supplementation. Improvement in the defense mechanism was noticed from the strengthened peritrophic membrane of the digestive tract and the increased total protein. Overall, the results presented illustrate that single cell protein-synthesized silver nanoparticles supplementation is effective in controlling viral-borne diseases of the silkworm.

  5. Bom m 9 from Bombyx mori is a novel protein related to asthma.

    PubMed

    Zuo, Jianhong; Lei, Mingsheng; Yang, Rui; Liu, Zhigang

    2015-07-01

    The silkworm (Bombyx mori) can cause severe IgE-mediated allergic disease, however, the mechanism remains unclear. The aim of this study was to investigate the immunologic mechanism by which silkworms induce allergy. Whole silkworm pupa proteins were separated by SDS-PAGE and 2-D PAGE. Then, IgE-binding proteins were detected by immunoblotting with sera of patients having an allergy to Bombyx mori. After tryptic digestion, the peptides of IgE-binding proteins were analyzed by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry or tandem mass spectrometry. Database searches were used to identify allergens in silkworm pupa, after which Bom m 9 was to construct an asthma model. Thus, in the current study, a mouse asthma model was constructed with Bom m 9.

  6. Evolutionarily Conserved Repulsive Guidance Role of Slit in the Silkworm Bombyx mori

    PubMed Central

    Liu, Chun; Cui, Wei-Zheng; Mu, Zhi-Mei; Zhao, Xiao; Liu, Qing-Xin

    2014-01-01

    Axon guidance molecule Slit is critical for the axon repulsion in neural tissues, which is evolutionarily conserved from planarians to humans. However, the function of Slit in the silkworm Bombyx mori was unknown. Here we showed that the structure of Bombyx mori Slit (BmSlit) was different from that in most other species in its C-terminal sequence. BmSlit was localized in the midline glial cell, the neuropil, the tendon cell, the muscle and the silk gland and colocalized with BmRobo1 in the neuropil, the muscle and the silk gland. Knock-down of Bmslit by RNA interference (RNAi) resulted in abnormal development of axons and muscles. Our results suggest that BmSlit has a repulsive role in axon guidance and muscle migration. Moreover, the localization of BmSlit in the silk gland argues for its important function in the development of the silk gland. PMID:25285792

  7. Characterization of Bombyx mori parvo-like virus non-structural protein NS1.

    PubMed

    Li, Guohui; Sun, Chen; Zhang, Junhong; He, Yuanqing; Chen, Huiqing; Kong, Jie; Huang, Guoping; Chen, Keping; Yao, Qin

    2009-12-01

    NS1 gene of Bombyx mori parvo-like virus (China Zhenjiang isolate, BmDNV-Z) codes a predicted 316-amino acid protein, but its function remains unknown. Results of the current study showed that purified recombinant 6 x His-NS1 protein possesses ATP binding, ATPase, DNA binding, and helicase activities. Only one protein was captured in infected Bombyx mori midgut cells against NS1 target protein by employing co-immunoprecipitation, which was identified to be a viral protein by mass spectrometry. The NS1-interacting protein is encoded by BmDNV-Z ORF4 and its molecular is about 100 kD. Analysis of His pull-down confirmed that binding of identified viral protein to purified recombinant 6 x His-NS1 protein in vitro. Taken together, our results indicated that BmDNV-Z NS1 was a multifunctional protein, which may be involved with virus replication.

  8. Embryonic thermosensitive TRPA1 determines transgenerational diapause phenotype of the silkworm, Bombyx mori.

    PubMed

    Sato, Azusa; Sokabe, Takaaki; Kashio, Makiko; Yasukochi, Yuji; Tominaga, Makoto; Shiomi, Kunihiro

    2014-04-01

    In the bivoltine strain of the silkworm, Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother; however, its molecular mechanisms are largely unknown. Here, we show that the Bombyx TRPA1 ortholog (BmTrpA1) acts as a thermosensitive transient receptor potential (TRP) channel that is activated at temperatures above ∼ 21 °C and affects the induction of diapause in progeny. In addition, we show that embryonic RNAi of BmTrpA1 affects diapause hormone release during pupal-adult development. This study identifying a thermosensitive TRP channel that acts as a molecular switch for a relatively long-term predictive adaptive response by inducing an alternative phenotype to seasonal polyphenism is unique.

  9. Chitin in the silk gland ducts of the spider Nephila edulis and the silkworm Bombyx mori.

    PubMed

    Davies, Gwilym J G; Knight, David P; Vollrath, Fritz

    2013-01-01

    Here we report the detection and localisation of chitin in the cuticle of the spinning ducts of both the spider Nephila edulis and the silkworm Bombyx mori. Our observations demonstrate that the duct walls of both animals contain chitin notwithstanding totally independent evolutionary pathways of the systems. We conclude that chitin may well be an essential component for the construction of spinning ducts; we further conclude that in both species chitin may indicate the evolutionary origin of the spinning ducts.

  10. Effect of Wheat Flour Noodles with Bombyx mori Powder on Glycemic Response in Healthy Subjects

    PubMed Central

    Suk, Wanhee; Kim, JiEun; Kim, Do-Yeon; Lim, Hyunjung; Choue, Ryowon

    2016-01-01

    Recent trial results suggest that the consumption of a low glycemic index (GI) diet is beneficial in the prevention of high blood glucose levels. Identifying active hypoglycemic substances in ordinary foods could be a significant benefit to the management of blood glucose. It has been hypothesized that noodles with Bombyx mori powder are a low GI food. We evaluated GI and changes in postprandial glucose levels following consumption of those noodles and compared them with those following consumption of plain wheat flour noodles (control) and glucose (reference) in healthy subjects. Thirteen males (age: 34.2±4.5 years, body mass index: 23.2±1.1 kg/m2) consumed 75 g carbohydrate portions of glucose and the 2 kinds of noodle after an overnight fast. Capillary blood was measured at time 0 (fasting), 15, 30, 45, 60, 90, 120, and 180 min from the start of each food intake. The GI values were calculated by taking the ratio of the incremental area under the blood glucose response curve (IAUC) for the noodles and glucose. There was a significant difference in postprandial glucose concentrations at 30 and 45 min between the control noodles and the noodles with Bombyx mori powder: the IAUC and GI for the noodles with Bombyx mori powder were significantly lower than those for glucose and plain wheat flour noodles. The wheat flour noodles with Bombyx mori powder could help prevent an increase in postprandial glucose response and possibly provide an alternative to other carbohydrate staple foods for glycemic management. PMID:27752491

  11. Effect of Wheat Flour Noodles with Bombyx mori Powder on Glycemic Response in Healthy Subjects.

    PubMed

    Suk, Wanhee; Kim, JiEun; Kim, Do-Yeon; Lim, Hyunjung; Choue, Ryowon

    2016-09-01

    Recent trial results suggest that the consumption of a low glycemic index (GI) diet is beneficial in the prevention of high blood glucose levels. Identifying active hypoglycemic substances in ordinary foods could be a significant benefit to the management of blood glucose. It has been hypothesized that noodles with Bombyx mori powder are a low GI food. We evaluated GI and changes in postprandial glucose levels following consumption of those noodles and compared them with those following consumption of plain wheat flour noodles (control) and glucose (reference) in healthy subjects. Thirteen males (age: 34.2±4.5 years, body mass index: 23.2±1.1 kg/m(2)) consumed 75 g carbohydrate portions of glucose and the 2 kinds of noodle after an overnight fast. Capillary blood was measured at time 0 (fasting), 15, 30, 45, 60, 90, 120, and 180 min from the start of each food intake. The GI values were calculated by taking the ratio of the incremental area under the blood glucose response curve (IAUC) for the noodles and glucose. There was a significant difference in postprandial glucose concentrations at 30 and 45 min between the control noodles and the noodles with Bombyx mori powder: the IAUC and GI for the noodles with Bombyx mori powder were significantly lower than those for glucose and plain wheat flour noodles. The wheat flour noodles with Bombyx mori powder could help prevent an increase in postprandial glucose response and possibly provide an alternative to other carbohydrate staple foods for glycemic management.

  12. In silico identification of novel chitinase-like proteins in the silkworm, Bombyx mori, genome.

    PubMed

    Pan, Ye; Lü, Peng; Wang, Yong; Yin, Lijing; Ma, Hexiang; Ma, Guohong; Chen, Keping; He, Yuanqing

    2012-01-01

    In insects, chitinases participate in the periodic shedding of old exoskeletons and the turnover of peritrophic membranes. Chitinase family members have been identified in dozens of species, including Tribolium castaneum, Drosophila melanogaster, and Anopheles gambiae. In this study, nine chitinases and three hypothetical chitinases have been identified in Bombyx mori L. (Lepidoptera: Bombycidae) through genome-wide searching. Phylogenetic analyses revealed that seven of them belong to the seven chitinase groups, respectively. BmCht25 and BmCht26 could not be grouped into the known chitinase groups, and might belong to two new groups of the chitinase family. BmCht10, BmCht25, and BmIDGF have glutamate amino acid substitutions in the active catalytic domain. Only BmCht5 and BmCht10 contain CBD domain and PEST sequences (rich in proline, glutamic acid, serine, and threonine). BmCht5 and BmCht26 are located on chromosome 7, and others (BmCht6, BmCht7, BmCht10, BmCht11, BmCht20, BmIDGF) are located on separate chromosomes of Bombyx mori, respectively. The present study provides important background information for future studies using Bombyx mori as a model organism for insect development and virus and host interaction.

  13. [Cloning and structural analysis of MLP in the silkworm, Bombyx mori].

    PubMed

    Liu, Yan; Niu, Bao-Long; Weng, Hong-Biao; Shen, Wei-Feng; He, Li-Hua; Qi, Xiao-Peng; Meng, Zhi-Qi

    2007-09-01

    The LIM domain is found in a wide variety of eukaryotic proteins that regulate gene expression and cell differentiation during development. Muscle LIM protein (MLP) gene in Bombyx mori has been cloned by blasting its EST database and PCR test in present report. The resulting sequence covers 2 327 bp of cDNA (GenBank accession No. DQ311195). It has a complete open reading fragment and encodes a 494 amino acid protein. Genomic DNA sequence contains 11 exons and 10 introns, with intron splicing following the GT-AG rule. M.W. and PI of the predicted MLP in Bombyx mori are 53.03 kDa and 8.29 respectively. A single LIM domain linked to a glyscine-rich region is found in a previously deposited LIM protein (AAR23823) in Bombyx mori. MLP identified in this report encodes a protein with five tandem LIM-glycine modules. The two LIM proteins could be produced by alternative splicing and both are probably involved in muscle cell differentiation. This work provides foundation for further research on the in vivo function of MLP.

  14. In silico Identification of Novel Chitinase-Like Proteins in the Silkworm, Bombyx mori, Genome

    PubMed Central

    Pan, Ye; Lü, Peng; Wang, Yong; Yin, Lijing; Ma, Hexiang; Ma, Guohong; Chen, Keping; He, Yuanqing

    2012-01-01

    In insects, chitinases participate in the periodic shedding of old exoskeletons and the turnover of peritrophic membranes. Chitinase family members have been identified in dozens of species, including Tribolium castaneum, Drosophila melanogaster, and Anopheles gambiae. In this study, nine chitinases and three hypothetical chitinases have been identified in Bombyx mori L. (Lepidoptera: Bombycidae) through genome-wide searching. Phylogenetic analyses revealed that seven of them belong to the seven chitinase groups, respectively. BmCht25 and BmCht26 could not be grouped into the known chitinase groups, and might belong to two new groups of the chitinase family. BmCht10, BmCht25, and BmIDGF have glutamate amino acid substitutions in the active catalytic domain. Only BmCht5 and BmCht10 contain CBD domain and PEST sequences (rich in proline, glutamic acid, serine, and threonine). BmCht5 and BmCht26 are located on chromosome 7, and others (BmCht6, BmCht7, BmCht10, BmCht11, BmCht20, BmIDGF) are located on separate chromosomes of Bombyx mori, respectively. The present study provides important background information for future studies using Bombyx mori as a model organism for insect development and virus and host interaction. PMID:23461297

  15. Structural study of Bombyx mori silk fibroin during processing for regeneration

    NASA Astrophysics Data System (ADS)

    Ha, Sung-Won

    Bombyx mori silk fibroin has excellent mechanical properties combined with flexibility, tissue compatibility, and high oxygen permeability in the wet condition. This important material should be dissolved and regenerated to be utilized as useful forms such as gel, film, fiber, powder, or non-woven. However, it has long been a problem that the regenerated fibroin materials show poor mechanical properties and brittleness. These problems were technically solved by improving a fiber processing method reported here. The regenerated fibroin fibers showed much better mechanical properties compared to the original silk fibers. This improved technique for the fiber processing of Bombyx mori silk fibroin may be used as a model system for other semi-crystalline fiber forming proteins, becoming available through biotechnology. The physical and chemical properties of the regenerated fibers were characterized by SinTechRTM tensile testing, X-ray diffraction, solid state 13C NMR spectroscopy, and SEM. Unlike synthetic polymers, the molecular weight distribution of Bombyx mori silk fibroin is mono-disperse because silk fibroin is synthesized from DNA template. Genetic studies have revealed the entire amino acid sequence of Bombyx mori silk fibroin. It is known that the crystalline silk II structure is composed of hexa-amino acid sequences, GAGAGS. However, in the amino acid sequence of Bombyx mori silk fibroin heavy chain, there are present 11 chemically irregular but evolutionarily conserved sequences with about 31 amino acid residues (irregular GT˜GT sequences). The structure and role of these irregular sequences have remained unknown. One of the most frequently appearing irregular sequences was synthesized by a peptide synthesizer. The three-dimensional structure of this irregular silk peptide was studied by the high resolution two-dimensional NMR technique. The three-dimensional structure of this peptide shows that it makes a turn or loop structure (distorted O shape), which

  16. Titanium dioxide nanoparticles relieve silk gland damage and increase cocooning of Bombyx mori under phoxim-induced toxicity.

    PubMed

    Li, Bing; Yu, Xiaohong; Gui, Suxin; Xie, Yi; Hong, Jie; Zhao, Xiaoyang; Sheng, Lei; Sang, Xuezi; Sun, Qingqing; Wang, Ling; Shen, Weide; Hong, Fashui

    2013-12-18

    Organophosphate pesticides are applied widely in the world for agricultural purposes, and their exposures often resulted in non-cocooning of Bombyx mori in China. TiO2 nanoparticles have been demonstrated to increase pesticide resistance of Bombyx mori. While the toxicity of phoxim is well-documented, very limited information exists on the mechanisms of TiO2 nanoparticles improving the cocooning function of Bombyx mori following exposure to phoxim. The present study was, therefore, undertaken to determine whether TiO2 nanoparticles attenuate silk gland injury and elevate cocooning of B. mori following exposure to phoxim. The findings suggested that phoxim exposure resulted in severe damages of the silk gland structure and significantly decreased the cocooning in the silk gland of Bombyx mori. Furthermore, phoxim exposure significantly resulted in reductions of total protein concentrations and suppressed expressions of silk protein synthesis-related genes, including Fib-L, Fib-H, P25, Ser-2, and Ser-3, in the silk gland. TiO2 nanoparticle pretreatment, however, could significantly relieve silk gland injury of Bombyx mori. Importantly, TiO2 nanoparticles could remarkably elevate cocooning and total protein contents and promote expressions of Fib-L, Fib-H, P25, Ser-2, and Ser-3 in the silk gland following exposure to phoxim.

  17. Chinese Americans.

    ERIC Educational Resources Information Center

    Lyman, Stanford M.

    This book on the Chinese Americans focuses on such aspects of intergroup relations, community characteristics, social problems, acculturation, racial and social discrimination, and economic opportunities for the ethnic group as: the Chinese diaspora; forerunners of overseas Chinese community organization; Chinese community organization in the…

  18. Juvenile hormone stimulated tyrosine kinase-mediated protein phosphorylation in the CNS of the silk worm, Bombyx mori.

    PubMed

    Arif, A; Shanavas, A; Murthy, Ch R K; Dutta-Gupta, Aparna

    2002-07-01

    In vitro studies with the larval CNS of the silkworm, Bombyx mori revealed the phosphorylation of a 48-kDa protein, which was not dependent on cyclic nucleotides. Studies also revealed modest phosphorylation of this protein by a calcium-dependent but calmodulin-independent mechanism. However, phosphorylation of this protein was greatly enhanced in the presence of juvenile hormone (JH) I by a calcium-independent mechanism. This stimulatory effect of JH was seen in both homogenates as well as in intact CNS of Bombyx. Immunoblotting studies revealed the cross-reaction of this 48-kDa protein with phosphotyrosine monoclonal antibody and the phosphorylation of this protein was inhibited by genistein. This study suggests that the 48-kDa protein is a substrate for tyrosine kinase. The phosphorylation of this protein was also observed in other larval tissues such as salivary gland, fat body, and epidermis of Bombyx.

  19. A midgut-specific serine protease, BmSP36, is involved in dietary protein digestion in the silkworm, Bombyx mori.

    PubMed

    Liu, Hua-Wei; Li, You-Shan; Tang, Xin; Guo, Peng-Chao; Wang, Dan-Dan; Zhou, Chun-Yan; Xia, Qing-You; Zhao, Ping

    2017-10-01

    Serine proteases play important roles in digestion and immune responses during insect development. In the present study, the serine protease gene BmSP36, which encodes a 292-residue protein, was cloned from the midgut cells of Bombyx mori. BmSP36 contains an intact catalytic triad (H57, D102 and S195) and a conserved substrate-binding site (G189, H216 and G226), suggesting that it is a serine protease with chymotrypsin-like specificity. The temporal and spatial expression patterns of BmSP36 indicated that its messenger RNA and protein expression mainly occurred in the midgut at the feeding stages. Western blotting, immunofluorescence and liquid chromatography-tandem mass spectrometry analyses revealed secretion of BmSP36 protein from epithelial cells into the midgut lumen. The transcriptional and translational expression of BmSP36 was down-regulated after starvation but up-regulated after refeeding. Moreover, expression of the BmSP36 gene could be up-regulated by a juvenile hormone analogue. These results enable us to better define the potential role of BmSP36 in dietary protein digestion at the feeding stages during larval development. © 2016 Institute of Zoology, Chinese Academy of Sciences.

  20. The clustered regularly interspaced short palindromic repeats/associated proteins system for the induction of gene mutations and phenotypic changes in Bombyx mori.

    PubMed

    Song, Jia; Che, Jiaqian; You, Zhengying; Ye, Xiaogang; Li, Jisheng; Ye, Lupeng; Zhang, Yuyu; Qian, Qiujie; Zhong, Boxiong

    2016-12-01

    To probe the general phenomena of gene mutations, Bombyx mori, the lepidopterous model organism, was chosen as the experimental model. To easily detect phenotypic variations, the piggyBac system was utilized to introduce two marker genes into the silkworm, and 23.4% transposition efficiency aided in easily breeding a new strain for the entire experiment. Then, the clustered regularly interspaced short palindromic repeats/an associated protein (Cas9) system was utilized. The results showed that the Cas9 system can induce efficient gene mutations and the base changes could be detected since the G0 individuals in B. mori; and that the mutation rates on different target sites were diverse. Next, the gRNA2-targeted site that generated higher mutation rate was chosen, and the experimental results were enumerated. First, the mutation proportion in G1 generation was 30.1%, and some gene mutations were not inherited from the G0 generation; second, occasionally, base substitutions did not lead to variation in the amino-acid sequence, which decreased the efficiency of phenotypic changes compared with that of genotypic changes. These results laid the foundation for better use of the Cas9 system in silkworm gene editing. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Characterization of a novel chromodomain-containing gene from the silkworm, Bombyx mori.

    PubMed

    Shoji, Keisuke; Kiuchi, Takashi; Hara, Kahori; Kawamoto, Munetaka; Kawaoka, Shinpei; Arimura, Shin-Ichi; Tsutsumi, Nobuhiro; Sugano, Sumio; Suzuki, Yutaka; Shimada, Toru; Katsuma, Susumu

    2013-09-25

    Heterochromatin protein 1 (HP1) is an evolutionarily conserved protein across different eukaryotic species, and is crucial in the establishment and maintenance of heterochromatin. HP1 proteins have two distinct functional domains, an N-terminal chromodomain (CD) and a C-terminal chromoshadow domain (CSD), which are required for the selective binding of HP1 proteins to modified histones. During our screen for HP1-like proteins in the Bombyx mori genome, we found a novel silkworm gene, Bombyx mori chromodomain protein 1 (BmCdp1), encoding a putative chromobox protein with only two CDs. The BmCdp1 family proteins are closely related to the HP1 proteins, and most of them belong to insect lineages. qRT-PCR analysis indicated that BmCdp1 mRNA was most abundantly expressed in early embryos, and relatively higher expression was observed in larval testes, hemocytes, and pupal ovaries. Western blot and immunostaining experiments showed that BmCdp1 was localized mainly in the nucleus of BmN4 cells. We searched BmCdp1-bound loci in the Bombyx genome by ChIP-seq analysis using Flag-tagged BmCdp1-expressing BmN4 cells. Combined with ChIP-qPCR experiments, we identified two reliable BmCdp1-bound loci in the genome. siRNA-mediated knockdown of BmCdp1 in BmN4 cells and early embryos did not affect the expression of the gene located close to the BmCdp1-bound locus.

  2. The angiotensin-converting enzyme (ACE) gene family of Bombyx mori.

    PubMed

    Yan, Hai-Yan; Mita, Kazuei; Zhao, Xia; Tanaka, Yoshikazu; Moriyama, Minoru; Wang, Huabin; Iwanaga, Masashi; Kawasaki, Hideki

    2017-04-15

    We previously reported regarding an ecdysone-inducible angiotensin-converting enzyme (ACE) gene. We found another four ACE genes in the Bombyx genome. The present study was undertaken to clarify the evolutionally changed function of the ACE of Bombyx mori. Core regions of deduced amino acid sequences of ACE genes were compared with those of other insect ACE genes. Five Bombyx genes have the conserved Zn(2+)-binding-site motif (HEXXH); however, BmAcer4 has only one and BmAcer3 has no catalytic ligand. BmAcer1 and BmAcer2 were expressed in several organs. BmAcer3 was expressed in testes, and BmAcer4 and BmAcer5 were expressed in compound eyes; however, the transcription levels of these three genes were very low. Quantitative RT-PCR and Western analysis were conducted to determine the tissue distribution and developmental expression of BmAcer1and BmAcer2. Transcripts of BmAcer1 and BmAcer2 were found in the reproductive organs during the larval and pupal stages. BmAcer1 was dominant in fat bodies during the feeding stage and showed high expression in the epidermis, wing discs, and pupal wing tissues after the wandering stage. Its expression patterns in epidermis, wing discs, and wing tissues resembled the hemolymph ecdysteroid titer in the larval and pupal stages. Acer1 was observed in the hemolymph at all stages, appearing to be the source of it are fat bodies, wings, and epidermis, and functioning after being secreted into the hemolymph. BmAcer2 was abundant in the midgut during the feeding stage and after the wandering stage and in silk glands after the pupal stage. We conclude that the evolution of BmAcer occurred through duplication, and, thereafter, functional diversification developed.

  3. Systematic cloning and analysis of autophagy-related genes from the silkworm Bombyx mori.

    PubMed

    Zhang, Xuan; Hu, Zhan-Ying; Li, Wei-Fang; Li, Qing-Rong; Deng, Xiao-Juan; Yang, Wan-Ying; Cao, Yang; Zhou, Cong-Zhao

    2009-05-27

    Through the whole life of eukaryotes, autophagy plays an important role in various biological events including development, differentiation and determination of lifespan. A full set of genes and their encoded proteins of this evolutionarily conserved pathway have been identified in many eukaryotic organisms from yeast to mammals. However, this pathway in the insect model organism, the silkworm Bombyx mori, remains poorly investigated. Based on the autophagy pathway in several model organisms and a series of bioinformatic analyses, we have found more than 20 autophagy-related genes from the current database of the silkworm Bombyx mori. These genes could be further classified into the signal transduction pathway and two ubiquitin-like pathways. Using the mRNA extracted from the silkgland, we cloned the full length cDNA fragments of some key genes via reverse transcription PCR and 3' rapid amplification of cDNA ends (RACE). In addition, we found that the transcription levels of two indicator genes BmATG8 and BmATG12 in the silkgland tend to be increased from 1st to 8th day of the fifth instar larvae. Bioinformatics in combination with RT-PCR enable us to remodel a preliminary pathway of autophagy in the silkworm. Amplification and cloning of most autophagy-related genes from the silkgland indicated autophagy is indeed an activated process. Furthermore, the time-course transcriptional profiles of BmATG8 and BmATG12 revealed that both genes are up-regulated along the maturation of the silkgland during the fifth instar. These findings suggest that the autophagy should play an important role in Bombyx mori silkgland.

  4. From genome to proteome: great progress in the domesticated silkworm (Bombyx mori L.).

    PubMed

    Zhou, Zhonghua; Yang, Huijuan; Zhong, Boxiong

    2008-07-01

    As the only truly domesticated insect, the silkworm not only has great economic value, but it also has value as a model for genetics and molecular biology research. Genomics and proteomics have recently shown vast potential to be essential tools in domesticated silkworm research, especially after the completion of the Bombyx mori genome sequence. This paper reviews the progress of the domesticated silkworm genome, particularly focusing on its genetic map, physical map and functional genome. This review also presents proteomics, the proteomic technique and its application in silkworm research.

  5. Entry of Bombyx mori nucleopolyhedrovirus into BmN cells by cholesterol-dependent macropinocytic endocytosis.

    PubMed

    Huang, Jinshan; Hao, Bifang; Cheng, Chen; Liang, Fei; Shen, Xingjia; Cheng, Xiaowen

    2014-10-10

    Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious viral pathogen of silkworm, and no drug or specific protection against BmNPV infection is available at present time. Although functions of most BmNPV genes were depicted in recent years, knowledge on the mechanism of BmNPV entry into insect cells is still limited. Here BmNPV cell entry mechanism is investigated by different endocytic inhibitor application and subcellular analysis. Results indicated that BmNPV enters BmN cells by clathrin-independent macropinocytic endocytosis, which is mediated by cholesterol in a dose-dependent manner, and cholesterol replenishment rescued the BmNPV infection partially.

  6. Structural and thermal properties of γ – irradiated Bombyx mori silk fibroin films

    SciTech Connect

    Madhukumar, R.; Asha, S.; Rao, B. Lakshmeesha; Shivananda, C. S.; Harish, K. V.; Sangappa; Sarojini, B. K.; Somashekar, R.

    2015-06-24

    The gamma radiation-induced change in structural and thermal properties of Bombyx mori silk fibroin films were investigated and have been correlated with the applied radiation doses. Irradiation of samples were carried out in dry air at room temperature using Co-60 source, and radiation doses are in the range of 0 - 300 kGy. Structural and thermal properties of the irradiated silk films were studied using X-ray diffraction (XRD), Differential Scanning Calorimetry (DSC) and Thermogravimetric analysis (TGA) and compared with unirradiated sample. Interesting results are discussed in this report.

  7. Identification of ommin in the integument of the silkworm, Bombyx mori.

    PubMed

    Sawada, H; Tsusué, M; Iino, T

    1994-06-01

    An ommin was isolated from the integument of the quail mutant of the silkworm, Bombyx mori, using column chromatography on SP-Sephadex and cellulose powder. As a standard, ommin was isolated from diapause eggs of the normal silkworm. The red pigment from the larval integument of quail mutants was identical to the standard compound with respect to absorption spectrum, infrared spectrum and RF values in thin-layer chromatography (TLC). After acid hydrolysis of the pigment, 3-hydroxykynurenine was detected by TLC. This is the first report of an ommin in a lepidopteran larval integument.

  8. Effects of BmKIT 3 R gene transfer on pupal development of Bombyx mori Linnaeus using a Gal4/UAS binary transgenic system.

    PubMed

    Zhang, Hao-Kun; Cao, Guang-Li; Zhang, Xiao-Rong; Wang, Xiao-Juan; Xue, Ren-Yu; Gong, Cheng-Liang

    2012-03-28

    The pupal stage of the silkworm Bombyx mori Linnaeus lasts for approximately two weeks. However, prolongation of pupal duration would reduce the labor required to process and dry fresh cocoons. This study investigated the effects of BmKIT(3)(R) gene (from the Chinese scorpion Buthus martensii Karsch) transfer on the pupal development of B. mori using a Gal4/UAS binary transgenic system. Gal4 driven by a pupa-specific promoter BmWCP4 (from a B. mori wing-cuticle protein gene) or PDP (from a B. mori cocoonase gene), and BmKIT(3)(R) driven by a UAS cis-acting element were used to construct novel piggyBac-derived plasmids containing a neomycin-resistance gene (neo) controlled by the Bombyx mori nucleopolyhedrovirus (BmNPV) ie-1 (immediate-early gene) promoter and a green fluorescent protein gene (gfp) under the control of the B. mori actin 3 (A3) promoter. The vector was transferred into silkworm eggs by sperm-mediated gene transfer. Transgenic silkworms were produced after screening for neo and gfp genes, and gene transfer was verified by polymerase chain reaction and dot-blot hybridization. The larval development of the hybrid progeny of Gal4- and UAS-transgenic silkworms was similar to that of normal silkworms, but some pupae failed to metamorphose into moths, and the development of surviving pupae was arrested as a result of BmKIT(3)(R) expression. Moreover, Gal4 driven by the BmWCP4 promoter delayed pupal development more effectively than that driven by the PDP promoter in the Gal4/UAS binary transgenic system. Pupal durations of hybrid transgenic silkworm progeny with BmWCP4 and PDP promoters were approximately 5, 2, and 4 days longer, respectively, compared to corresponding normal silkworms, BmWCP4/Gal4, and UAS/BmKIT(3)(R) transgenic silkworms, respectively. These results suggest new avenues of research for prolonging the pupal duration of silkworms.

  9. Heritable genome editing with CRISPR/Cas9 in the silkworm, Bombyx mori.

    PubMed

    Wei, Wei; Xin, Huhu; Roy, Bhaskar; Dai, Junbiao; Miao, Yungen; Gao, Guanjun

    2014-01-01

    We report the establishment of an efficient and heritable gene mutagenesis method in the silkworm Bombyx mori using modified type II clustered regularly interspaced short palindromic repeats (CRISPR) with an associated protein (Cas9) system. Using four loci Bm-ok, BmKMO, BmTH, and Bmtan as candidates, we proved that genome alterations at specific sites could be induced by direct microinjection of specific guide RNA and Cas9-mRNA into silkworm embryos. Mutation frequencies of 16.7-35.0% were observed in the injected generation, and DNA fragments deletions were also noted. Bm-ok mosaic mutants were used to test for mutant heritability due to the easily determined translucent epidermal phenotype of Bm-ok-disrupted cells. Two crossing strategies were used. In the first, injected Bm-ok moths were crossed with wild-type moths, and a 28.6% frequency of germline mutation transmission was observed. In the second strategy, two Bm-ok mosaic mutant moths were crossed with each other, and 93.6% of the offsprings appeared mutations in both alleles of Bm-ok gene (compound heterozygous). In summary, the CRISPR/Cas9 system can act as a highly specific and heritable gene-editing tool in Bombyx mori.

  10. Identification and quantification and antioxidant activity of flavonoids in different strains of silk cocoon, Bombyx mori.

    PubMed

    Napavichayanun, Supamas; Lutz, Oliver; Fischnaller, Martin; Jakschitz, Thomas; Bonn, Günther; Aramwit, Pornanong

    2017-10-01

    Silk cocoon is produced from silkworm (Bombyx mori) to protect itself from outer environment. Various strains of cocoon contain different forms and amounts of flavonoids, which may affect on their antioxidant activity. Moreover, the extraction method would influence the amount of flavonoids extracted. Therefore, the objectives of this study were to identify and quantify the flavonoids in 3 strains of bivoltine Bombyx mori silk cocoon (Chul 1/1; white cocoon, Chul 3/2; greenish cocoon, and Chul 4/2; yellow cocoon) extracted by 6 different solvents including acetone, ethyl acetate, dimethyl sulfoxide (DMSO), ethanol, methanol, and purified water. The flavonoids extracted were identified and quantified by liquid chromatography-mass spectrometry (LC-MS). The antioxidant activity of flavonoids extracted was also investigated by visible spectroscopy at 517 nm. The results showed that Chul 3/2 silk cocoon contained the highest amount of flavonoids. Purified water seemed to be the best solvent that preserved most antioxidant activity of the flavonoids extracted. Flavonoids in Chul 1/1 and Chul 4/2 silk cocoon were rarely found, however they contained some antioxidant activities. The data from this study can provide basic information for flavonoid extraction from silk cocoon which can also apply for other flavonoid-containing natural biomaterials. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Identification and analysis of YELLOW protein family genes in the silkworm, Bombyx mori

    PubMed Central

    Xia, Ai-Hua; Zhou, Qing-Xiang; Yu, Lin-Lin; Li, Wei-Guo; Yi, Yong-Zhu; Zhang, Yao-Zhou; Zhang, Zhi-Fang

    2006-01-01

    Background The major royal jelly proteins/yellow (MRJP/YELLOW) family possesses several physiological and chemical functions in the development of Apis mellifera and Drosophila melanogaster. Each protein of the family has a conserved domain named MRJP. However, there is no report of MRJP/YELLOW family proteins in the Lepidoptera. Results Using the YELLOW protein sequence in Drosophila melanogaster to BLAST silkworm EST database, we found a gene family composed of seven members with a conserved MRJP domain each and named it YELLOW protein family of Bombyx mori. We completed the cDNA sequences with RACE method. The protein of each member possesses a MRJP domain and a putative cleavable signal peptide consisting of a hydrophobic sequence. In view of genetic evolution, the whole Bm YELLOW protein family composes a monophyletic group, which is distinctly separate from Drosophila melanogaster and Apis mellifera. We then showed the tissue expression profiles of Bm YELLOW protein family genes by RT-PCR. Conclusion A Bombyx mori YELLOW protein family is found to be composed of at least seven members. The low homogeneity and unique pattern of gene expression by each member among the family ensure us to prophesy that the members of Bm YELLOW protein family would play some important physiological functions in silkworm development. PMID:16884544

  12. Identification and Analysis of the SET-Domain Family in Silkworm, Bombyx mori.

    PubMed

    Zhao, Hailong; Zheng, Chunqin; Cui, Hongjuan

    2015-01-01

    As an important economic insect, Bombyx mori is also a useful model organism for lepidopteran insect. SET-domain-containing proteins belong to a group of enzymes named after a common domain that utilizes the cofactor S-adenosyl-L-methionine (SAM) to achieve methylation of its substrates. Many SET-domain-containing proteins have been shown to display catalytic activity towards particular lysine residues on histones, but emerging evidence also indicates that various nonhistone proteins are specifically targeted by this clade of enzymes. To explore their diverse functions of SET-domain superfamily in insect, we identified, cloned, and analyzed the SET-domains proteins in silkworm, Bombyx mori. Firstly, 24 genes containing SET domain from silkworm genome were characterized and 17 of them belonged to six subfamilies of SUV39, SET1, SET2, SUV4-20, EZ, and SMYD. Secondly, SET domains of silkworm SET-domain family were intraspecifically and interspecifically conserved, especially for the catalytic core "NHSC" motif, substrate binding site, and catalytic site in the SET domain. Lastly, further analyses indicated that silkworm SET-domain gene BmSu(var)3-9 owned different characterization and expression profiles compared to other invertebrates. Overall, our results provide a new insight into the functional and evolutionary features of SET-domain family.

  13. The expression profile and promoter analysis of ultraspiracle gene in the silkworm Bombyx mori.

    PubMed

    Huang, Ming-xia; Du, Jie; Su, Bao-jin; Zhao, Guo-dong; Shen, Wei-de; Wei, Zheng-guo

    2014-12-01

    The nuclear receptor, ultraspiracle protein (USP), is a transcription factor and an essential component of a heterodimeric receptor complex with ecdysone receptor. However, the mechanisms underlying the transcriptional regulation of USP in silkworm are unknown. In this study, using dual-spike-in qPCR method, we examined the expression of Bombyx ultraspiracle gene (BmUSP) in various tissues of silkworm as well as expression changes after stimulation with ecdysone. The results showed that the expression levels of BmUSP gene varied in different tissues and were increased 2 h after exposure to ecdysone. To identify the molecular mechanism underlying the regulation of USP gene expression in silkworm Bombyx mori, promoter truncation analyses were performed using the luciferase reporter assay and Bac-to-Bac expression system in several tissues of B. mori. BmUSP gene promoter with 5' end serial deletions showed different levels of activity in various tissues, higher in fat body and Malpighian tubule. Deletion of the region from -485 to -445 and -307 to -281 upstream of BmUSP gene abolished and increased its promoter activity, respectively. This region contains AP-1, Dfd transcription factor binding sites. These results indicate that BmUSP are expressed at different levels in different tissues of the silkworm, but all are subjected to the regulation by ecdysone. This study would provide an important foundation for investigating the mechanism underlying the transcriptional regulation of BmUSP in the silkworm.

  14. Sublethal dose of phoxim and Bombyx mori nucleopolyhedrovirus interact to elevate silkworm mortality.

    PubMed

    Gu, ZhiYa; Li, FanChi; Hu, JingSheng; Ding, Chao; Wang, Chaoqian; Tian, JiangHai; Xue, Bin; Xu, KaiZun; Shen, WeiDe; Li, Bing

    2017-03-01

    Silkworm (Bombyx mori) is an economically important insect. It is relatively less resistant to certain chemicals and environment exposures such as pesticides and pathogens. After pesticide exposures, the silkworms are more susceptible to microbial infections. The mechanism underlying the susceptibility might be related to immune response and oxidative stress. A sublethal dose of phoxim combined with Bombyx mori nucleopolyhedrovirus (BmNPV) elevated the silkworm mortality at 96 h. We found a higher content of H2 O2 and increased levels of genes related to oxidative stress and immune response after treatment with a sublethal dose of phoxim for 24 h or 48 h. However, such response decreased with longer pesticide treatment. Mortality increased by 44% when B. mori was exposed to combined treatment with BmNPV and phoxim rather than BmNPV alone. The level of examined immune-related and oxidative-stress-related genes significantly decreased in the combined treatment group compared with the BmNPV group. Our results indicated that, with long-term exposure to pesticides such as OPs, even at sublethal dose, the oxidative stress response and immune responses in silkworm were inhibited, which may lead to further immune impairment and accumulation of oxidative stress, resulting in susceptibility to the virus and harm to the silkworm. Our study provided insights for understanding the susceptibility to pathogen after pesticide exposures, which may promote the development of better pesticide controls to avoid significant economic losses. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  15. Identification and Analysis of the SET-Domain Family in Silkworm, Bombyx mori

    PubMed Central

    Zhao, Hailong; Zheng, Chunqin; Cui, Hongjuan

    2015-01-01

    As an important economic insect, Bombyx mori is also a useful model organism for lepidopteran insect. SET-domain-containing proteins belong to a group of enzymes named after a common domain that utilizes the cofactor S-adenosyl-L-methionine (SAM) to achieve methylation of its substrates. Many SET-domain-containing proteins have been shown to display catalytic activity towards particular lysine residues on histones, but emerging evidence also indicates that various nonhistone proteins are specifically targeted by this clade of enzymes. To explore their diverse functions of SET-domain superfamily in insect, we identified, cloned, and analyzed the SET-domains proteins in silkworm, Bombyx mori. Firstly, 24 genes containing SET domain from silkworm genome were characterized and 17 of them belonged to six subfamilies of SUV39, SET1, SET2, SUV4-20, EZ, and SMYD. Secondly, SET domains of silkworm SET-domain family were intraspecifically and interspecifically conserved, especially for the catalytic core “NHSC” motif, substrate binding site, and catalytic site in the SET domain. Lastly, further analyses indicated that silkworm SET-domain gene BmSu(var)3-9 owned different characterization and expression profiles compared to other invertebrates. Overall, our results provide a new insight into the functional and evolutionary features of SET-domain family. PMID:26558257

  16. 20-hydroxyecdysone upregulates Atg genes to induce autophagy in the Bombyx fat body

    PubMed Central

    Tian, Ling; Ma, Li; Guo, Enen; Deng, Xiaojuan; Ma, Sanyuan; Xia, Qingyou; Cao, Yang; Li, Sheng

    2013-01-01

    Autophagy is finely regulated at multiple levels and plays crucial roles in development and disease. In the fat body of the silkworm, Bombyx mori, autophagy occurs and Atg gene expression peaks during the nonfeeding molting and pupation stages when the steroid hormone (20-hydroxyecdysone; 20E) is high. Injection of 20E into the feeding larvae upregulated Atg genes and reduced TORC1 activity resulting in autophagy induction in the fat body. Conversely, RNAi knockdown of the 20E receptor partner (USP) or targeted overexpression of a dominant negative mutant of the 20E receptor (EcRDN) in the larval fat body reduced autophagy and downregulated the Atg genes, confirming the importance of 20E-induction of Atg gene expression during pupation. Moreover, in vitro treatments of the larval fat body with 20E upregulated the Atg genes. Five Atg genes were potentially 20E primary-responsive, and a 20E response element was identified in the Atg1 (ortholog of human ULK1) promoter region. Furthermore, RNAi knockdown of 4 key genes (namely Br-C, E74, HR3 and βftz-F1) in the 20E-triggered transcriptional cascade reduced autophagy and downregulated Atg genes to different levels. Taken together, we conclude that in addition to blocking TORC1 activity for autophagosome initiation, 20E upregulates Atg genes to induce autophagy in the Bombyx fat body. PMID:23674061

  17. Interaction of Bombyx mori nucleopolyhedrovirus BRO-A and host cell protein laminin.

    PubMed

    Kang, W K; Imai, N; Suzuki, M; Iwanaga, M; Matsumoto, S; Zemskov, E A

    2003-01-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) contains five baculovirus repeated ORF ( bro) genes, all of which are expressed as delayed early genes. We have recently reported that BmNPV BRO proteins, specially BRO-A and BRO-C, contain a nucleic acid binding activity and are involved in nucleosome structures in nuclei of infected cells. To further understand the function of bro-a gene, we looked for factors interacting with BmNPV BRO-A using the yeast two-hybrid system. Fifteen clones obtained from a cDNA library of mock-infected cells and one from a library prepared at 2 h postinfection (p.i.) were found to comprise one distinct gene, which was identified as the Bombyx homolog (bLaminin) of Drosophila laminin beta1. A direct interaction between BRO-A and N-terminal region of bLaminin was demonstrated by in vitro pull-down experiments. Further pull-down assays using BmN cell extracts and anti-laminin antibodies also showed interaction of both proteins. In addition, two more clones were obtained from cDNA library of 12 h p.i. and were found to encode BRO-A itself, indicating that BRO-A forms an oligomer. Taken together, we propose that BRO-A may function as a laminin binding protein.

  18. Isozymic variations in specific and nonspecific esterase and its thermostability in silkworm, Bombyx mori L.

    PubMed

    Patnaik, Bharat Bhusan; Biswas, Tapati Datta; Nayak, Sandeepta Kumar; Saha, A K; Majumdar, M K

    2012-09-01

    Esterase isozymic variations were documented in the haemolymph of developed multivoltine and bivoltine silkworm breeds during unfavorable seed crop seasons of May - September using á- and â- napthylacetate separately to identify specific and nonspecific esterase having thermotolerant potentiality. Variations existed in the isozyme pattern with three bands (Est-2, 3 and 4) in pure Nistari race and other developed multivoltine and bivoltine breeds. Est-2 and Est-3 were non-specific esterases as they were observed when both á- and â-napthylacetate was used as substrates separately. Est-4 band was observed only with á-napthylacetate as substrate and was therefore confirmed to be specific á-esterase band in the haemolymph of silkworm, Bombyx mori L. Zymograms showed that the non-specific esterase band (Est-3) with R1 of 0.43 and specific á-esterase band (Est-4) with R(f) of 0.32 predominately withstood a temperature of 70 +/- 2 degrees C for a duration of 10 min and were confirmed as thermostable esterases in haemolymph of silkworm, Bombyx mori L. This also categorized the presence of thermostable esterases in developed multivoltine and bivoltine breeds of silkworm, even though the qualitative activity was more in the former than the latter. The qualitative presence of thermostable esterases and their activity could be adopted as an indicative biochemical marker in relation to thermotolerance in silkworm.

  19. 20-Hydroxyecdysone upregulates Atg genes to induce autophagy in the Bombyx fat body.

    PubMed

    Tian, Ling; Ma, Li; Guo, Enen; Deng, Xiaojuan; Ma, Sanyuan; Xia, Qingyou; Cao, Yang; Li, Sheng

    2013-08-01

    Autophagy is finely regulated at multiple levels and plays crucial roles in development and disease. In the fat body of the silkworm, Bombyx mori, autophagy occurs and Atg gene expression peaks during the nonfeeding molting and pupation stages when the steroid hormone (20-hydroxyecdysone; 20E) is high. Injection of 20E into the feeding larvae upregulated Atg genes and reduced TORC1 activity resulting in autophagy induction in the fat body. Conversely, RNAi knockdown of the 20E receptor partner (USP) or targeted overexpression of a dominant negative mutant of the 20E receptor (EcR (DN) ) in the larval fat body reduced autophagy and downregulated the Atg genes, confirming the importance of 20E-induction of Atg gene expression during pupation. Moreover, in vitro treatments of the larval fat body with 20E upregulated the Atg genes. Five Atg genes were potentially 20E primary-responsive, and a 20E response element was identified in the Atg1 (ortholog of human ULK1) promoter region. Furthermore, RNAi knockdown of 4 key genes (namely Br-C, E74, HR3 and βftz-F1) in the 20E-triggered transcriptional cascade reduced autophagy and downregulated Atg genes to different levels. Taken together, we conclude that in addition to blocking TORC1 activity for autophagosome initiation, 20E upregulates Atg genes to induce autophagy in the Bombyx fat body.

  20. Cells of the rectum of Bombyx mori affected by experimental inoculation with Alphabaculovirus.

    PubMed

    Vessaro-Silva, S A; Brancalhão, R M C; Baggio, M P D; Ribeiro, L F C

    2014-08-29

    Bombyx mori is an insect whose cocoon is used in the sericulture industry, which is an important activity in parts of southern Brazil. When parasitized by Bombyx mori nucleopolyhedrovirus (BmNPV) of the genus Alphabaculovirus (alphaBV), it develops nuclear polyhedrosis disease. In Brazil, an alphaBV was isolated from larvae of B. mori and various target tissues were identified. However, how this geographic viral isolate affects the rectum of silk moths was unknown. The rectum, a component of the cryptonephric system, acts to absorb water and mineral salts, and its importance for the metabolic balance of insects provoked interest in analyzing how it is affected by BmMNPV infection. Fifth instar B. mori larvae were inoculated with a viral suspension, and from the second to the ninth day post-inoculation, segments of the rectum (anterior and anal canal) were examined using light microscopy. The cryptonephric epithelial cells in the anterior region revealed no evidence of infection. However, from the fifth day post-inoculation, cells of the anal canal showed cytopathologies characteristic of alphaBV. Infection of the anal canal and other surrounding tissues led to tissue disorganization, with accumulation of polyhedra in the perinephric space and compartmentalization of the cryptonephric system, promoting changes in the fecal pellets, signalling physiological changes. These observations contribute to our understanding of the infectious cycle of BmMNPV in B. mori.

  1. Transcriptional analysis of the DNA polymerase gene of Bombyx mori parvo-like virus (China isolate).

    PubMed

    Wang, Yong-Jie; Chen, Ke-Ping; Yao, Qin; Han, Xu

    2007-04-01

    The Bombyx mori parvo-like virus (China isolate) DNA polymerase (BmDNV-3 dnapol) gene has been tentatively identified based on the presence of conserved motifs. In the present study, we perform a transcriptional analysis of the BmDNV-3 dnapol gene using the total RNA isolated from BmDNV-3 infected silkworm at different times. Northern blot analysis with a BmDNV-3 dnapol-specific riboprobe showed a major transcript of 3.3 kb. 5'-RACE revealed that the major transcription start point was located 20 nucleotides downstream of the TATA box. In a temporal expression analysis using differential RT-PCR, BmDNV-3 dnapol transcript was detected at low levels at 6 h.p.i., increased from 6 to 36 h.p.i., and remained fairly constant thereafter. Analysis of the predicted DNA polymerase sequence using neighborjoining and protein parsimony algorithms indicated that the predicted 1115-residue polypeptide contained five motifs associated with DNA polymerases synthetic activities and three additional motifs associated with polymerases possessing 3' to 5' exonuclease activity. The molecular phylogenetic analysis of this gene supported the placement of Bombyx mori parvo-like virus in a separate virus family.

  2. Surface Display and Bioactivity of Bombyx mori Acetylcholinesterase on Pichia pastoris

    PubMed Central

    He, Yong-Sheng; Beier, Ross C.; Sun, Yuan-Ming; Xu, Zhen-Lin; Wu, Wei-Jian; Shen, Yu-Dong; Xiao, Zhi-Li; Lai, Li-Na; Wang, Hong; Yang, Jin-Yi

    2013-01-01

    A Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE) was constructed and its bioactivity was studied. The modified Bombyx mori acetylcholinesterase gene (bmace) was fused with the anchor protein (AGα1) from Saccharomyces cerevisiae and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGα1 was induced to display BmAChE on the P. pastoris cell surface. Fluorescence microscopy and flow cytometry assays revealed that the BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of the displayed BmAChE was detected by the Ellman method at 787.7 U/g (wet cell weight). In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine, and with carbamate and organophosphorus pesticides. The displayed BmAChE had an IC50 of 4.17×10−8 M and was highly sensitive to eserine and five carbamate pesticides, as well as seven organophosphorus pesticides. Results suggest that the displayed BmAChE had good bioactivity. PMID:23940577

  3. Characterization of Bombyx mori mitochondrial transcription factor A, a conserved regulator of mitochondrial DNA.

    PubMed

    Sumitani, Megumi; Kondo, Mari; Kasashima, Katsumi; Endo, Hitoshi; Nakamura, Kaoru; Misawa, Toshihiko; Tanaka, Hiromitsu; Sezutsu, Hideki

    2017-04-15

    In the present study, we initially cloned and characterized a mitochondrial transcription factor A (Tfam) homologue in the silkworm, Bombyx mori. Bombyx mori TFAM (BmTFAM) localized to mitochondria in cultured silkworm and human cells, and co-localized with mtDNA nucleoids in human HeLa cells. In an immunoprecipitation analysis, BmTFAM was found to associate with human mtDNA in mitochondria, indicating its feature as a non-specific DNA-binding protein. In spite of the low identity between BmTFAM and human TFAM (26.5%), the expression of BmTFAM rescued mtDNA copy number reductions and enlarged mtDNA nucleoids in HeLa cells, which were induced by human Tfam knockdown. Thus, BmTFAM compensates for the function of human TFAM in HeLa cells, demonstrating that the mitochondrial function of TFAM is highly conserved between silkworms and humans. BmTfam mRNA was strongly expressed in early embryos. Through double-stranded RNA (dsRNA)-based RNA interference (RNAi) in silkworm embryos, we found that the knockdown of BmTFAM reduced the amount of mtDNA and induced growth retardation at the larval stage. Collectively, these results demonstrate that BmTFAM is a highly conserved mtDNA regulator and may be a good candidate for investigating and modulating mtDNA metabolism in this model organism. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Surface display and bioactivity of Bombyx mori acetylcholinesterase on Pichia pastoris.

    PubMed

    Dong, Jie-Xian; Xie, Xi; He, Yong-Sheng; Beier, Ross C; Sun, Yuan-Ming; Xu, Zhen-Lin; Wu, Wei-Jian; Shen, Yu-Dong; Xiao, Zhi-Li; Lai, Li-Na; Wang, Hong; Yang, Jin-Yi

    2013-01-01

    A Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE) was constructed and its bioactivity was studied. The modified Bombyx mori acetylcholinesterase gene (bmace) was fused with the anchor protein (AGα1) from Saccharomyces cerevisiae and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGα1 was induced to display BmAChE on the P. pastoris cell surface. Fluorescence microscopy and flow cytometry assays revealed that the BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of the displayed BmAChE was detected by the Ellman method at 787.7 U/g (wet cell weight). In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine, and with carbamate and organophosphorus pesticides. The displayed BmAChE had an IC50 of 4.17×10(-8) M and was highly sensitive to eserine and five carbamate pesticides, as well as seven organophosphorus pesticides. Results suggest that the displayed BmAChE had good bioactivity.

  5. Novel non-autonomous transposable elements on W chromosome of the silkworm, Bombyx mori.

    PubMed

    Abe, Hiroaki; Fujii, Tsuguru; Shimada, Toru; Mita, Kazuei

    2010-09-01

    The sex chromosomes of the silkworm Bombyx mori are designated ZW(XY) for females and ZZ (XX) for males. Numerous long terminal repeat (LTR) and non-LTR retrotransposons, retroposons and DNA transposons have accumulated as strata on the W chromosome. However, there are nucleotide sequences that do not show the characteristics of typical transposable elements on the W chromosome. To analyse these uncharacterized nucleotide sequences on the W chromosome, we used whole-genome shotgun (WGS) data and assembled data that was obtained using male genome DNA. Through these analyses,we found that almost all of these uncharacterized sequences were non-autonomous transposable elements that do not fit into the conventional classification. It is notable that some of these transposable elements contained the Bombyx short interspersed element (Bm1) sequences in the elements. We designated them as secondary-Bm1 transposable elements (SBTEs). Because putative ancestral SBTE nucleotide sequences without Bm1 do not occur in the WGS data, we suggest that the Bm1 sequences of SBTEs are not carried on each element merely as a package but are components of each element. Therefore, we confirmed that SBTEs should be classified as a new group of transposable elements.

  6. Novel non-autonomous transposable elements on W chromosome of the silkworm, Bombyx mori.

    PubMed

    Abe, Hiroaki; Fujii, Tsuguru; Shimada, Toru; Mita, Kazuei

    2010-01-01

    The sex chromosomes of the silkworm Bombyx mori are designated ZW (XY) for females and ZZ (XX) for males. Numerous long terminal repeat (LTR) and non-LTR retrotransposons, retroposons and DNA transposons have accumulated as strata on the W chromosome. However, there are nucleotide sequences that do not show the characteristics of typical transposable elements on the W chromosome. To analyse these uncharacterized nucleotide sequences on the W chromosome, we used whole-genome shotgun (WGS) data and assembled data that was obtained using male genome DNA. Through these analyses, we found that almost all of these uncharacterized sequences were non-autonomous transposable elements that do not fit into the conventional classification. It is notable that some of these transposable elements contained the Bombyx short interspersed element (Bm1) sequences in the elements. We designated them as secondary-Bm1 transposable elements (SBTEs). Because putative ancestral SBTE nucleotide sequences without Bm1 do not occur in theWGS data, we suggest that the Bm1 sequences of SBTEs are not carried on each element merely as a package but are components of each element. Therefore, we confirmed that SBTEs should be classified as a new group of transposable elements.

  7. Bombyx mori E26 transformation-specific 2 (BmEts2), an Ets family protein, represses Bombyx mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in the silkworm Bombyx mori.

    PubMed

    Tanaka, H; Sagisaka, A; Suzuki, N; Yamakawa, M

    2016-10-01

    E26 transformation-specific (Ets) family transcription factors are known to play roles in various biological phenomena, including immunity, in vertebrates. However, the mechanisms by which Ets proteins contribute to immunity in invertebrates remain poorly understood. In this study, we identified a cDNA encoding BmEts2, which is a putative orthologue of Drosophila Yan and human translocation-ets-leukemia/Ets-variant gene 6, from the silkworm Bombyx mori. Expression of the BmEts2 gene was significantly increased in the fat bodies of silkworm larvae in response to injection with Escherichia coli and Staphylococcus aureus. BmEts2 overexpression dramatically repressed B. mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in silkworm cells. Conversely, gene knockdown of BmEts2 significantly enhanced BmRels activity. In addition, two κB sites located on the 5' upstream region of cecropin B1 were found to be involved in the repression of BmRels-mediated promoter activation. Protein-competition analysis further demonstrated that BmEts2 competitively inhibited binding of BmRels to κB sites. Overall, BmEts2 acts as a repressor of BmRels-mediated transactivation of antimicrobial protein genes by inhibiting the binding of BmRels to κB sites. © 2016 The Royal Entomological Society.

  8. Analyses of interactions among pair-rule genes and the gap gene Krüppel in Bombyx segmentation.

    PubMed

    Nakao, Hajime

    2015-09-01

    In the short-germ insect Tribolium, a pair-rule gene circuit consisting of the Tribolium homologs of even-skipped, runt, and odd-skipped (Tc-eve, Tc-run and Tc-odd, respectively) has been implicated in segment formation. To examine the application of the model to other taxa, I studied the expression and function of pair-rule genes in Bombyx mori, together with a Bombyx homolog of Krüppel (Bm-Kr), a known gap gene. Knockdown embryos of Bombyx homologs of eve, run and odd (Bm-eve, Bm-run and Bm-odd) exhibited asegmental phenotypes similar to those of Tribolium knockdowns. However, pair-rule gene interactions were similar to those of both Tribolium and Drosophila, which, different from Tribolium, shows a hierarchical segmentation mode. Additionally, the Bm-odd expression pattern shares characteristics with those of Drosophila pair-rule genes that receive upstream regulatory input. On the other hand, Bm-Kr knockdowns exhibited a large posterior segment deletion as observed in short-germ insects. However, a detailed analysis of these embryos indicated that Bm-Kr modulates expression of pair-rule genes like in Drosophila, although the mechanisms appear to be different. This suggested hierarchical interactions between Bm-Kr and pair-rule genes. Based on these results, I concluded that the pair-rule gene circuit model that describes Tribolium development is not applicable to Bombyx.

  9. MicroRNA-281 regulates the expression of ecdysone receptor (EcR) isoform B in the silkworm, Bombyx mori

    USDA-ARS?s Scientific Manuscript database

    Hundreds of Bombyx mori miRNAs had been identified in recent years, but their function in vivo remains poorly understood. The silkworm EcR gene (BmEcR) has three transcriptional isoforms, A, B1 and B2. Isoform sequences are different in the 3’UTR region of the gene, which is the case only in insects...

  10. Identification and Functional Characterization of Two Orphan G-protein-coupled Receptors for Adipokinetic Hormones from Silkworm Bombyx mori*

    PubMed Central

    Shi, Ying; Huang, Haishan; Deng, Xiaoyan; He, Xiaobai; Yang, Jingwen; Yang, Huipeng; Shi, Liangen; Mei, Lijuan; Gao, Jimin; Zhou, Naiming

    2011-01-01

    Adipokinetic hormones (AKHs) are the best studied insect neuropeptides with the function of mobilizing lipids and carbohydrates during energy-expensive activities and modulating fundamental physiological processes, such as sugar homeostasis, lipid metabolism, and reproduction. Three distinct cDNAs encoding the prepro-Bombyx AKH1–3 have been cloned and confirmed by mass spectrometric methods. Our previous research suggested the Bombyx AKH receptor is activated by AKH1 and AKH2 with high affinity but by AKH3 with quite low affinity. In this study, using stable functional expression of the receptors in HEK293 cells, we have now identified AKH3 as a specific ligand for two orphan G-protein-coupled receptors, and we therefore named them AKHR2a and AKHR2b, respectively. We demonstrated that both AKHR2a and AKHR2b were activated by AKH3 at high affinity and by AKH1 and AKH2 at low affinity, leading to an increase of intracellular cAMP levels and activation of ERK1/2 and receptor internalization, but they were not activated by Bombyx corazonin. Conversely, the Bombyx corazonin receptor was activated by corazonin but not by AKH1–3. Quantitative RT-PCR revealed that AKHR2a and AKHR2b were both highly expressed in the testis but were also detected at low levels in other tissues. These results will lead to a better understanding of the AKH/AKHR system in the regulation of fundamental physiological processes. PMID:22009754

  11. Chinese Literature

    ERIC Educational Resources Information Center

    Hsu, Kai-yu

    The earliest recorded Chinese literature that has survived consists of folk songs mixed with verses and rhymes. Two factors determined the general pattern of subsequent development in Chinese literature: the nature of the written Chinese language and the establishment of the Confucian school as the orthodoxy in literary criticism. By 1800 there…

  12. Chinese Literature

    ERIC Educational Resources Information Center

    Hsu, Kai-yu

    The earliest recorded Chinese literature that has survived consists of folk songs mixed with verses and rhymes. Two factors determined the general pattern of subsequent development in Chinese literature: the nature of the written Chinese language and the establishment of the Confucian school as the orthodoxy in literary criticism. By 1800 there…

  13. Characterization of an entomopathogenic fungi target integument protein, Bombyx mori single domain von Willebrand factor type C, in the silkworm, Bombyx mori.

    PubMed

    Han, F; Lu, A; Yuan, Y; Huang, W; Beerntsen, B T; Huang, J; Ling, E

    2017-06-01

    The insect cuticle works as the first line of defence to protect insects from pathogenic infections and water evaporation. However, the old cuticle must be shed in order to enter the next developmental stage. During each ecdysis, moulting fluids are produced and secreted into the area among the old and new cuticles. In a previous study, the protein Bombyx mori single domain von Willebrand factor type C (BmSVWC; BGIBMGA011399) was identified in the moulting fluids of Bo. mori and demonstrated to regulate ecdysis. In this study we show that in Bo. mori larvae, BmSVWC primarily locates to the integument (epidermal cells and cuticle), wing discs and head. During the moulting stage, BmSVWC is released into the moulting fluids, and is then produced again by epidermal cells after ecdysis. Fungal infection was shown to decrease the amount of BmSVWC in the cuticle, which indicates that BmSVWC is a target protein of entomopathogenic fungi. Thus, BmSVWC is mainly involved in maintaining the integrity of the integument structure, which serves to protect insects from physical damage and pathogenic infection. © 2017 The Royal Entomological Society.

  14. Advances in silkworm studies accelerated by the genome sequencing of Bombyx mori.

    PubMed

    Xia, Qingyou; Li, Sheng; Feng, Qili

    2014-01-01

    Significant progress has been achieved in silkworm (Bombyx mori) research since the last review on this insect was published in this journal in 2005. In this article, we review the new and exciting progress and discoveries that have been made in B. mori during the past 10 years, which include the construction of a fine genome sequence and a genetic variation map, the evolution of genomes, the advent of functional genomics, the genetic basis of silk production, metamorphic development, immune response, and the advances in genetic manipulation. These advances, which were accelerated by the genome sequencing project, have promoted B. mori as a model organism not only for lepidopterans but also for general biology.

  15. Vitellogenin from the Silkworm, Bombyx mori: An Effective Anti-Bacterial Agent

    PubMed Central

    Kumar, Manish; Prasad, Tulika; Kannan, Mani; König, Simone

    2013-01-01

    Silkworm, Bombyx mori, vitellogenin (Vg) was isolated from perivisceral fat body of day 3 of pupa. Both Vg subunits were co-purified as verified by mass spectrometry and immunoblot. Purified Vg responded to specific tests for major posttranslational modifications on native gels indicating its nature as lipo-glyco-phosphoprotein. The Vg fraction had strong antibacterial activity against Gram negative bacterium Escherichia coli and Gram positive bacterium Bacillus subtilis. Microscopic images showed binding of Vg to bacterial cells and their destruction. When infected silkworm larvae were treated with purified Vg they survived the full life cycle in contrast to untreated animals. This result showed that Vg has the ability to inhibit the proliferation of bacteria in the silkworm fluid system without disturbing the regular metabolism of the host. PMID:24058454

  16. Microstructural, thermal and antibacterial properties of electron beam irradiated Bombyx mori silk fibroin films

    SciTech Connect

    Asha, S.; Sanjeev, Ganesh; Sangappa; Naik, Prashantha; Chandra, K. Sharat

    2014-04-24

    The Bombyx mori silk fibroin (SF) films were prepared by solution casting method and the effects of electron beam on structural, thermal and antibacterial responses of the prepared films were studied. The electron irradiation for different doses was carried out using 8 MeV Microtron facility at Mangalore University. The changes in microstructural parameters and thermal stability of the films were investigated using Wide Angle X-ray Scattering (WAXS) and thermogravimetric analysis (TGA) respectively. Both microstructuralline parameters (crystallite size and lattice strain (g in %)) and thermal stability of the irradiated films have increased with radiation dosage. Agar diffusion method demonstrated the antibacterial activity of SF film which was increased after irradiation on both Gram-positive and Gram-negative species.

  17. Identification of a diazinon-metabolizing glutathione S-transferase in the silkworm, Bombyx mori

    PubMed Central

    Yamamoto, Kohji; Yamada, Naotaka

    2016-01-01

    The glutathione S-transferase superfamily play key roles in the metabolism of numerous xenobiotics. We report herein the identification and characterization of a novel glutathione S-transferase in the silkworm, Bombyx mori. The enzyme (bmGSTu2) conjugates glutathione to 1-chloro-2,4-dinitrobenzene, as well as metabolizing diazinon, one of the organophosphate insecticides. Quantitative reverse transcription–polymerase chain reaction analysis of transcripts demonstrated that bmGSTu2 expression was induced 1.7-fold in a resistant strain of B. mori. Mutagenesis of putative amino acid residues in the glutathione-binding site revealed that Ile54, Glu66, Ser67, and Asn68 are crucial for enzymatic function. These results provide insights into the catalysis of glutathione conjugation in silkworm by bmGSTu2 and into the detoxification of organophosphate insecticides. PMID:27440377

  18. Optical properties of γ-irradiated Bombyx mori silk fibroin films

    NASA Astrophysics Data System (ADS)

    Madhukumar, R.; Asha, S.; Lakshmeesha Rao, B.; Sarojini, B. K.; Byrappa, K.; Wang, Youjiang; Sangappa, Y.

    2015-11-01

    In the present work the Bombyx mori silk fibroin (SF) films were prepared by the solution casting method and effects of γ-irradiation on the optical properties and optical constants of the films have been studied by using Ultra Violet-Visible (UV-Vis) spectrophotometer. The recorded UV-Vis absorption and transmission spectra have been used to determine the optical band gap (Eg), refractive index (n), extinction coefficient (k), optical conductivity (σopt) and dielectric constants (ε*) of virgin and γ-irradiated films. Reduction in optical band gap and increase in refractive index with increasing radiation dosage were observed. It is also found that there is an increase in dielectric constants with increasing photon energy. The obtained results reveal that the refractive index of the SF films may be efficiently changed by γ-irradiation.

  19. Dissolution and regeneration of Bombyx mori silk fibroin using ionic liquids.

    PubMed

    Phillips, David M; Drummy, Lawrence F; Conrady, Deborah G; Fox, Douglas M; Naik, Rajesh R; Stone, Morley O; Trulove, Paul C; De Long, Hugh C; Mantz, Robert A

    2004-11-10

    In this work, the suitability of imidazolium-based ionic liquid solvents is investigated for the dissolution and regeneration of silkworm (Bombyx mori) silk. Within an ionic liquid the anion plays a larger role in dictating the ultimate solubility of the silk. The dissolution of the silk in the ionic liquid is confirmed using wide-angle X-ray scattering. The dissolved silk is also processed into 100 mum-thick, two-dimensional films, and the structure of these films is examined. The rinse solvent, acetonitrile or methanol, has a profound impact on both the topography of the films and the secondary structure of the silk protein. The image depicts a silkworm cocoon dissolved in 1-butyl-3-methylimidazolium chloride and then regenerated as a film with birefringence.

  20. Interactions between fibroin and sericin proteins from Antheraea pernyi and Bombyx mori silk fibers.

    PubMed

    Du, Shan; Zhang, Jin; Zhou, Wei T; Li, Quan X; Greene, George W; Zhu, Hai J; Li, Jing L; Wang, Xun G

    2016-09-15

    Silkworm silk fibers are core-shell composites of fibroin and sericin proteins. Studying the interactions between fibroin and sericin is essential for understanding the properties of these composites. It is observed that compared to the domestic silk cocoon Bombyx mori (B. mori), the adhesion between fibroin and sericin from the wild silk cocoon, Antheraea pernyi (A. pernyi), is significantly stronger with a higher degree of heterogeneity. The adsorption of A. pernyi sericin on its fibroin is almost twice the value for B. mori sericin on fibroin, both showing a monolayer Langmuir adsorption. (1)H NMR and FTIR studies demonstrate on a molecular level the stronger interactions and the more intensive complex formation between A. pernyi fibroin and sericin, facilitated by the hydrogen bonding between glycine and serine. The findings of this study may help the design of composites with superior interfacial adhesion between different components. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Effect of Venom from the Jellyfish Nemopilema nomurai on the Silkworm Bombyx mori L

    PubMed Central

    Yu, Huahua; Li, Rongfeng; Chen, Xiaolin; Yue, Yang; Xing, Ronge; Liu, Song; Li, Pengcheng

    2015-01-01

    The silkworm Bombyx mori L. (B. mori) has a significant impact on the economy by producing more than 80% of the globally produced raw silk. The exposure of silkworm to pesticides may cause adverse effects on B. mori, such as a reduction in the production and quality of silk. This study aims to assay the effect of venom from the jellyfish Nemopilema nomurai on growth, cuticle and acetylcholinesterase (AChE) activity of the silkworm B. mori by the leaf dipping method. The experimental results revealed that the four samples caused neither antifeeding nor a lethal effect on B. mori. The sample SFV inhibited B. mori growth after 6 days of treatment in a dose-dependent manner. The samples SFV, DSFV and Fr-1 inhibited the precipitation and synthesis of chitin in the cuticle after 12 and 14 days of treatment. In the case of the four samples, the AChE was significantly improved after 14 days of treatment. PMID:26404374

  2. Biochemical properties of an omega-class glutathione S-transferase of the silkmoth, Bombyx mori.

    PubMed

    Yamamoto, Kohji; Nagaoka, Sumiharu; Banno, Yutaka; Aso, Yoichi

    2009-05-01

    A cDNA encoding an omega-class glutathione S-transferase of the silkmoth, Bombyx mori (bmGSTO), was cloned by reverse transcriptase-polymerase chain reaction. The resulting clone was sequenced and deduced for amino acid sequence, which revealed 40, 40, and 39% identities to omega-class GSTs from human, pig, and mouse, respectively. A recombinant protein (rbmGSTO) was functionally overexpressed in Escherichia coli cells in a soluble form and purified to homogeneity. rbmGSTO was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a model substrate for GST, as well as with 4-hydroxynonenal, a product of lipid peroxidation. This enzyme was shown to have high affinity for organophosphorus insecticide and was present abundantly in silkmoth strain exhibiting fenitrothion resistance. These results indicate that bmGSTO could be involved in the increase in level of insecticide resistance for lepidopteran insects.

  3. BmECM25, from the silkworm Bombyx mori, is an extracellular matrix protein.

    PubMed

    Zou, Ziliang; Xu, Yunmin; Ma, Bi; Xiang, Zhonghuai; He, Ningjia

    2015-10-01

    BmECM25 (previously reported as BmVMP25) was previously predicted as a gene encoding the vitelline membrane protein in silkworm, Bombyx mori. In this study, we investigated the detail temporal and spatial patterns of BmECM25 protein. Western blot results showed that BmECM25 was expressed in the follicular epithelium cells from stages -6 to +1, and was then secreted into the oocytes. However, the abundance of BmECM25 decreased during the subsequent oogenesis and finally disappeared in the mature follicles. Immunofluorescence detection showed that BmECM25 locates inside the VM layer and forms a discontinuous layer. These features of BmECM25 suggest that it is an oocyte membrane matrix protein, not a vitelline membrane protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. BmRobo2/3 is required for axon guidance in the silkworm Bombyx mori.

    PubMed

    Li, Xiao-Tong; Yu, Qi; Zhou, Qi-Sheng; Zhao, Xiao; Liu, Zhao-Yang; Cui, Wei-Zheng; Liu, Qing-Xin

    2016-02-15

    Axon guidance is critical for proper wiring of the nervous system. During the neural development, the axon guidance molecules play a key role and direct axons to choose the correct way to reach the target. Robo, as the receptor of axon guidance molecule Slit, is evolutionarily conserved from planarians to humans. However, the function of Robo in the silkworm, Bombyx mori, remained unknown. In this study, we cloned robo2/3 from B. mori (Bmrobo2/3), a homologue of robo2/3 in Tribolium castaneum. Moreover, BmRobo2/3 was localized in the neuropil, and RNAi-mediated knockdown of Bmrobo2/3 resulted in the longitudinal connectives forming closer to the midline. These data demonstrate that BmRobo2/3 is required for axon guidance in the silkworm. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Analysis of tissue-specific region in sericin 1 gene promoter of Bombyx mori

    SciTech Connect

    Liu Yan; Yu Lian; Guo Xiuyang; Guo Tingqing; Wang Shengpeng; Lu Changde . E-mail: cdlu@sibs.ac.cn

    2006-03-31

    The gene encoding sericin 1 (Ser1) of silkworm (Bombyx mori) is specifically expressed in the middle silk gland cells. To identify element involved in this transcription-dependent spatial restriction, truncation of the 5' terminal from the sericin 1 (Ser1) promoter is studied in vivo. A 209 bp DNA sequence upstream of the transcriptional start site (-586 to -378) is found to be responsible for promoting tissue-specific transcription. Analysis of this 209 bp region by overlapping deletion studies showed that a 25 bp region (-500 to -476) suppresses the ectopic expression of the Ser1 promoter. An unknown factor abundant in fat body nuclear extracts is shown to bind to this 25 bp fragment. These results suggest that this 25 bp region and the unknown factor are necessary for determining the tissue-specificity of the Ser1 promoter.

  6. The extramacrochaetae gene is required for blastokinesis in silkworm, Bombyx mori.

    PubMed

    Liu, Wenbin; Chai, Dezhi; Wang, Cailian; Li, Qing; Lei, Jinfeng; Yang, Min; Dai, Fangyin; Lu, Cheng

    2015-07-01

    In silkworm, Bombyx mori Linnaeus (Lepidoptera: Bombycidae), blastokinesis results in embryo reversal from ventrally to dorsally convex flexion. In this study, we showed that the extramacrochaetae (emc) gene is required for blastokinesis in silkworm. Depletion of Bmemc expression via RNA interference led to severe phenotypic defects in blastokinesis. The defective embryos failed to invert their body sides during blastokinesis. This caused the posterior half of the abdomen to abnormally fold back toward the dorsal side, forming a U-shaped morphology. Dorsal closure was also disrupted. Our results suggest that Bmemc is involved in blastokinesis of silkworm embryos. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 405-409, 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  7. Effect of Venom from the Jellyfish Nemopilema nomurai on the Silkworm Bombyx mori L.

    PubMed

    Yu, Huahua; Li, Rongfeng; Chen, Xiaolin; Yue, Yang; Xing, Ronge; Liu, Song; Li, Pengcheng

    2015-09-24

    The silkworm Bombyx mori L. (B. mori) has a significant impact on the economy by producing more than 80% of the globally produced raw silk. The exposure of silkworm to pesticides may cause adverse effects on B. mori, such as a reduction in the production and quality of silk. This study aims to assay the effect of venom from the jellyfish Nemopilema nomurai on growth, cuticle and acetylcholinesterase (AChE) activity of the silkworm B. mori by the leaf dipping method. The experimental results revealed that the four samples caused neither antifeeding nor a lethal effect on B. mori. The sample SFV inhibited B. mori growth after 6 days of treatment in a dose-dependent manner. The samples SFV, DSFV and Fr-1 inhibited the precipitation and synthesis of chitin in the cuticle after 12 and 14 days of treatment. In the case of the four samples, the AChE was significantly improved after 14 days of treatment.

  8. Transcription factor SGF1 is critical for the neurodevelopment in the silkworm, Bombyx mori.

    PubMed

    Liu, Zhao-Yang; Yu, Qi; Yang, Chun-Hong; Meng, Miao; Ren, Chun-Jiu; Mu, Zhi-Mei; Cui, Wei-Zheng; Liu, Qing-Xin

    2016-08-01

    FoxA transcription factors play vital roles in regulating the expression of organ-specific genes. BmSGF1, the sole FoxA family member in Bombyx mori, is required for development of the silk gland. However, the function of BmSGF1 in development of the nervous system in the silkworm remains unknown. Here, we show that the amino acids sequence of BmSGF1 is evolutionarily conserved in its middle region from Trichoplax adhaerens to human and diverged from the homologues in most other species in its N-terminal region. BmSGF1 expresses in the nervous system at the embryonic stage. Knockdown of Bmsgf1 by RNA interference (RNAi) results in abnormal development of axons. Therefore, our results demonstrate that BmSGF1 is an indispensable regulator for neurodevelopment. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Silkworm Gut Fiber of Bombyx mori as an Implantable and Biocompatible Light-Diffusing Fiber.

    PubMed

    Cenis, Jose Luis; Aznar-Cervantes, Salvador D; Lozano-Pérez, Antonio Abel; Rojo, Marta; Muñoz, Juan; Meseguer-Olmo, Luis; Arenas, Aurelio

    2016-07-16

    This work describes a new approach to the delivery of light in deeper tissues, through a silk filament that is implantable, biocompatible, and biodegradable. In the present work, silkworm gut fibers (SGFs) of Bombyx mori L., are made by stretching the silk glands. Morphological, structural, and optical properties of the fibers have been characterized and the stimulatory effect of red laser light diffused from the fiber was assayed in fibroblast cultures. SGFs are formed by silk fibroin (SF) mainly in a β-sheet conformation, a stable and non-soluble state in water or biological fluids. The fibers showed a high degree of transparency to visible and infrared radiation. Using a red laser (λ = 650 nm) as source, the light was efficiently diffused along the fiber wall, promoting a significant increment in the cell metabolism 5 h after the irradiation. SGFs have shown their excellent properties as light-diffusing optical fibers with a stimulatory effect on cells.

  10. Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N.

    PubMed

    Yaoi, K; Nakanishi, K; Kadotani, T; Imamura, M; Koizumi, N; Iwahana, H; Sato, R

    1999-12-17

    The Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N (APN) was analyzed, to better understand the molecular mechanism of susceptibility to the toxin and the development of resistance in insects. APN was digested with lysylendopeptidase and the ability of the resulting fragments to bind to Cry1Aa and 1Ac toxins was examined. The binding abilities of the two toxins to these fragments were different. The Cry1Aa toxin bound to the fragment containing 40-Asp to 313-Lys, suggesting that the Cry1Aa toxin-binding site is located in the region between 40-Asp and 313-Lys, while Cry1Ac toxin bound exclusively to mature APN. Next, recombinant APN of various lengths was expressed in Escherichia coli cells and its ability to bind to Cry1Aa toxin was examined. The results localized the Cry1Aa toxin binding to the region between 135-Ile and 198-Pro.

  11. Silkworm Gut Fiber of Bombyx mori as an Implantable and Biocompatible Light-Diffusing Fiber

    PubMed Central

    Cenis, Jose Luis; Aznar-Cervantes, Salvador D.; Lozano-Pérez, Antonio Abel; Rojo, Marta; Muñoz, Juan; Meseguer-Olmo, Luis; Arenas, Aurelio

    2016-01-01

    This work describes a new approach to the delivery of light in deeper tissues, through a silk filament that is implantable, biocompatible, and biodegradable. In the present work, silkworm gut fibers (SGFs) of Bombyx mori L., are made by stretching the silk glands. Morphological, structural, and optical properties of the fibers have been characterized and the stimulatory effect of red laser light diffused from the fiber was assayed in fibroblast cultures. SGFs are formed by silk fibroin (SF) mainly in a β-sheet conformation, a stable and non-soluble state in water or biological fluids. The fibers showed a high degree of transparency to visible and infrared radiation. Using a red laser (λ = 650 nm) as source, the light was efficiently diffused along the fiber wall, promoting a significant increment in the cell metabolism 5 h after the irradiation. SGFs have shown their excellent properties as light-diffusing optical fibers with a stimulatory effect on cells. PMID:27438824

  12. Structure and in vitro transcription of a glycine tRNA gene from Bombyx mori.

    PubMed Central

    Fournier, A; Guérin, M A; Corlet, J; Clarkson, S G

    1984-01-01

    We report the sequences of a Bombyx mori tRNA1Gly gene, its flanking regions, and its in vitro transcription products. The 5' flanking DNA contains the sequences TATAC, TATTTT and TTC located 30, 18 and 4 nucleotides, respectively, in front of the transcription initiation site. These resemble, in both position and composition, sequences preceding other RNA polymerase III genes of B. mori. A deletion mutant retaining these conserved sequences and an additional 8 bp of flanking DNA is transcribed better than the wild-type gene in cell-free extracts from Xenopus laevis and B. mori. A mutant lacking the conserved sequences is expressed in the frog extract, but is inactive in the homologous system. Images Fig. 3. Fig. 5. PMID:6745242

  13. Resistance to Bombyx mori nucleopolyhedrovirus via overexpression of an endogenous antiviral gene in transgenic silkworms.

    PubMed

    Jiang, Liang; Wang, Genhong; Cheng, Tingcai; Yang, Qiong; Jin, Shengkai; Lu, Gai; Wu, Fuquan; Xiao, Yang; Xu, Hanfu; Xia, Qingyou

    2012-07-01

    Transgenic technology is a powerful tool for improving disease-resistant species. Bmlipase-1, purified from the midgut juice of Bombyx mori, showed strong antiviral activity against B. mori nucleopolyhedrovirus (BmNPV). In an attempt to create an antiviral silkworm strain for sericulture, a transgenic vector overexpressing the Bmlipase-1 gene was constructed under the control of a baculoviral immediate early-1 (IE1) promoter. Transgenic lines were generated via embryo microinjection. The mRNA level of Bmlipase-1 in the midguts of the transgenic line was 27.3 % higher than that of the non-transgenic line. After feeding the silkworm with different amounts of BmNPV, the mortality of the transgenic line decreased to approximately 33 % compared with the non-transgenic line when the virus dose was 10(6) OB/larva. These results imply that overexpressing endogenous antiviral genes can enhance the antiviral resistance of silkworms.

  14. Transgene-based, female-specific lethality system for genetic sexing of the silkworm, Bombyx mori

    PubMed Central

    Tan, Anjiang; Fu, Guoliang; Jin, Li; Guo, Qiuhong; Li, Zhiqian; Niu, Baolong; Meng, Zhiqi; Morrison, Neil I.; Alphey, Luke; Huang, Yongping

    2013-01-01

    Transgene-based genetic sexing methods are being developed for insects of agricultural and public health importance. Male-only rearing has long been sought in sericulture because males show superior economic characteristics, such as better fitness, lower food consumption, and higher silk yield. Here we report the establishment of a transgene-based genetic sexing system for the silkworm, Bombyx mori. We developed a construct in which a positive feedback loop regulated by sex-specific alternative splicing leads to high-level expression of the tetracycline-repressible transactivator in females only. Transgenic animals show female-specific lethality during embryonic and early larval stages, leading to male-only cocoons. This transgene-based female-specific lethal system not only has wide application in sericulture, but also has great potential in lepidopteran pest control. PMID:23569267

  15. Transgene-based, female-specific lethality system for genetic sexing of the silkworm, Bombyx mori.

    PubMed

    Tan, Anjiang; Fu, Guoliang; Jin, Li; Guo, Qiuhong; Li, Zhiqian; Niu, Baolong; Meng, Zhiqi; Morrison, Neil I; Alphey, Luke; Huang, Yongping

    2013-04-23

    Transgene-based genetic sexing methods are being developed for insects of agricultural and public health importance. Male-only rearing has long been sought in sericulture because males show superior economic characteristics, such as better fitness, lower food consumption, and higher silk yield. Here we report the establishment of a transgene-based genetic sexing system for the silkworm, Bombyx mori. We developed a construct in which a positive feedback loop regulated by sex-specific alternative splicing leads to high-level expression of the tetracycline-repressible transactivator in females only. Transgenic animals show female-specific lethality during embryonic and early larval stages, leading to male-only cocoons. This transgene-based female-specific lethal system not only has wide application in sericulture, but also has great potential in lepidopteran pest control.

  16. Shotgun analysis on the peritrophic membrane of the silkworm Bombyx mori.

    PubMed

    Zhong, Xiaowu; Zhang, Liping; Zou, Yong; Yi, Qiying; Zhao, Ping; Xia, Qingyou; Xiang, Zhonghuai

    2012-11-01

    The insect midgut epithelium is generally lined with a unique chitin and protein structure, the peritrophic membrane (PM), which facilitates food digestion and protects the gut epithelium. We used gel electrophoresis and mass spectrometry to identify the extracted proteins from the silkworm PM to obtain an in-depth understanding of the biological function of the silkworm PM components. A total of 305 proteins, with molecular weights ranging from 8.02 kDa to 788.52 kDa and the isoelectric points ranging from 3.39 to 12.91, were successfully identified. We also found several major classes of PM proteins, i.e. PM chitin-binding protein, invertebrate intestinal mucin, and chitin deacetylase. The protein profile provides a basis for further study of the physiological events in the PM of Bombyx mori.

  17. Shotgun analysis on the peritrophic membrane of the silkworm Bombyx mori

    PubMed Central

    Zhong, Xiaowu; Zhang, Liping; Zou, Yong; Yi, Qiying; Zhao, Ping; Xia, Qingyou; Xiang, Zhonghuai

    2012-01-01

    The insect midgut epithelium is generally lined with a unique chitin and protein structure, the peritrophic membrane (PM), which facilitates food digestion and protects the gut epithelium. We used gel electrophoresis and mass spectrometry to identify the extracted proteins from the silkworm PM to obtain an in-depth understanding of the biological function of the silkworm PM components. A total of 305 proteins, with molecular weights ranging from 8.02 kDa to 788.52 kDa and the isoelectric points ranging from 3.39 to 12.91, were successfully identified. We also found several major classes of PM proteins, i.e. PM chitin-binding protein, invertebrate intestinal mucin, and chitin deacetylase. The protein profile provides a basis for further study of the physiological events in the PM of Bombyx mori. [BMB Reports 2012; 45(11): 665-670] PMID:23187007

  18. Production of antibacterial Bombyx mori cecropin A in mealworm-pathogenic Beauveria bassiana ERL1170.

    PubMed

    Lee, Se Jin; Yu, Jeong Seon; Parker, Bruce L; Skinner, Margaret; Je, Yeon Ho; Kim, Jae Su

    2015-01-01

    Efforts are underway to produce antimicrobial peptides in yellow mealworms (Tenebrio molitor), which can be developed as more effective and safer animal feed additives. In this work, we expressed Bombyx mori (Bm) cecropin-A in mealworms by the infection of transformed entomopathogenic Beauveria bassiana ERL1170. The active domain of Bm cecropin A gene was tagged with a signal sequence of B. bassiana for extracellular secretion, and the fragment was inserted into ERL1170 by the restriction enzyme-mediated integration method. Transformant D-6 showed antibacterial activity against Bacillus subtilis and Listeria monocytogenes. Against T. molitor larvae, D-6 had similar mortality to wild-type, and D6-infected mealworm suspension showed strong antibacterial activity against the two bacteria, but not in the wild-type-infected mealworms, thereby increasing the value of mealworms as animal feed additives.

  19. Forced reeling of Bombyx mori silk: separating behavior and processing conditions.

    PubMed

    Mortimer, Beth; Holland, Chris; Vollrath, Fritz

    2013-10-14

    Controlled reeling is a powerful tool to investigate the details of silk processing. However, consistent forced reeling of silkworms is hindered by the significant degree of behaviorally induced variation caused by the animal. This paper proposes silkworm paralysis as a novel method to control the animal and thus in vivo spinning conditions. Using these methods, we achieve low and consistent reeling forces during the collection of over 500 m of individual silk fiber while monitoring filament variability, morphology, and properties. Novel techniques to measure the irregular silk cross-sectional areas lead to the more accurate calculation of the true engineering values and mechanical property variation of individual silk fibers. Combining controlled reeling and accurate thread measurement techniques allows us to present the relative contributions of processing and behavior in the performance envelope of Bombyx mori silk.

  20. The advances and perspectives of recombinant protein production in the silk gland of silkworm Bombyx mori.

    PubMed

    Xu, Hanfu

    2014-10-01

    The silk gland of silkworm Bombyx mori, is one of the most important organs that has been fully studied and utilized so far. It contributes finest silk fibers to humankind. The silk gland has excellent ability of synthesizing silk proteins and is a kind tool to produce some useful recombinant proteins, which can be widely used in the biological, biotechnical and pharmaceutical application fields. It's a very active area to express recombinant proteins using the silk gland as a bioreactor, and great progress has been achieved recently. This review recapitulates the progress of producing recombinant proteins and silk-based biomaterials in the silk gland of silkworm in addition to the construction of expression systems. Current challenges and future trends in the production of valuable recombinant proteins using transgenic silkworms are also discussed.

  1. Silkworm pupae (Bombyx mori) are new sources of high quality protein and lipid.

    PubMed

    Tomotake, Hiroyuki; Katagiri, Mitsuaki; Yamato, Masayuki

    2010-01-01

    This study was performed to evaluate the nutritional value of silkworm pupae (Bombyx mori) and the content of α-glucosidase inhibitor. The percentages of total protein and lipid contents by dry weight were 55.6 and 32.2%, respectively. Silkworm pupae protein had high levels of essential amino acids such as valine, methionine and phenylalanine. The contents of essential amino acids in silkworm pupae protein satisfied the FAO/WHO/UNU suggested requirements (2007). In addition, they also possessed n-3 fatty acids, especially α-linolenic acid (36.3%), as a major component. The 50% ethanol extract of silkworm pupae contained 1-deoxynojirimycin (DNJ), which is a potent α-glucosidase inhibitor. These results suggest that silkworm pupae are a new source of high quality protein, lipid, and α-glucosidase inhibitor.

  2. Characterization and affinity purification of juvenile hormone esterase from Bombyx mori.

    PubMed

    Shiotsuki, T; Bonning, B C; Hirai, M; Kikuchi, K; Hammock, B D

    2000-08-01

    Juvenile hormone esterase (JHE) from hemolymph of the silkworm moth Bombyx mori was characterized for substrate specificity and inhibitor sensitivity. B. mori JHE hydrolyzed the juvenile hormone surrogate substrate methyl n-heptylthioacetothioate (HEPTAT) more efficiently than p-nitrophenyl acetate and 1-naphthyl acetate substrates widely used to assay total carboxylesterase activity. B. mori JHE was sensitive to 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP), which was developed as a selective inhibitor for lepidopteran JHE, and relatively insensitive to diisopropyl fluorophosphate (DFP), an inhibitor of serine esterases but not of all JHEs. Affinity purification with a trifluoromethyl ketone ligand was more efficient for purification of B. mori JHE than DEAE ion exchange chromatography.

  3. Identification and purification of a Bombyx mori homologue of FTZ-F1.

    PubMed Central

    Ueda, H; Hirose, S

    1990-01-01

    Extracts from embryos and from posterior and middle silk glands of the silkworm, Bombyx mori contain a sequence specific DNA binding factor termed BmFTZ-F1. The factor binds to the recognition site of FTZ-F1, a positive regulator of the fushi tarazu gene in Drosophila melanogaster. BmFTZ-F1 and FTZ-F1 share the same methylation interference patterns, the same chromatographic behaviors and similar protease digestion profiles. Anti-FTZ-F1 cross reacts with BmFTZ-F1. These results indicate that BmFTZ-F1 is a B. mori homologue of FTZ-F1. The mobility of the factor-DNA complex formed in the silk gland extract changes depending on the developmental stages. Purification of BmFTZF1 to an almost homogeneous state reveals that the factor is a 73 kd protein. Images PMID:2124348

  4. Analysis of expression and chitin-binding activity of the wing disc cuticle protein BmWCP4 in the silkworm, Bombyx mori.

    PubMed

    Deng, Hui-Min; Li, Yong; Zhang, Jia-Ling; Liu, Lin; Feng, Qi-Li

    2016-12-01

    The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin-binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic "R&R" chitin-binding domain (CBD). Western blotting showed that BmWCP4 was predominately expressed in the wing disc-containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full-length BmWCP4 protein, "R&R" CBD peptide (CBD), non-CBD peptide (BmWCP4-CBD(-) ), four single site-directed mutated peptides (M1 , M2 , M3 and M4 ) and four-sites-mutated peptide (MF ) were generated and purified, respectively, for in vitro chitin-binding assay. The results indicated that both the full-length protein and the "R&R" CBD peptide could bind with chitin, whereas the BmWCP4-CBD(-) could not bind with chitin. The single residue mutants M1 , M2 , M3 and M4 reduced but did not completely abolish the chitin-binding activity, while four-sites-mutated protein MF completely lost the chitin-binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva-to-pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein. © 2015 Institute of Zoology, Chinese Academy of Sciences.

  5. Genome-wide identification, characterization of sugar transporter genes in the silkworm Bombyx mori and role in Bombyx mori nucleopolyhedrovirus (BmNPV) infection.

    PubMed

    Govindaraj, Lekha; Gupta, Tania; Esvaran, Vijaya Gowri; Awasthi, Arvind Kumar; Ponnuvel, Kangayam M

    2016-04-01

    Sugar transporters play an essential role in controlling carbohydrate transport and are responsible for mediating the movement of sugars into cells. These genes exist as large multigene families within the insect genome. In insects, sugar transporters not only have a role in sugar transport, but may also act as receptors for virus entry. Genome-wide annotation of silkworm Bombyx mori (B. mori) revealed 100 putative sugar transporter (BmST) genes exists as a large multigene family and were classified into 11 sub families, through phylogenetic analysis. Chromosomes 27, 26 and 20 were found to possess the highest number of BmST paralogous genes, harboring 22, 7 and 6 genes, respectively. These genes occurred in clusters exhibiting the phenomenon of tandem gene duplication. The ovary, silk gland, hemocytes, midgut and malphigian tubules were the different tissues/cells enriched with BmST gene expression. The BmST gene BGIBMGA001498 had maximum EST transcripts of 134 and expressed exclusively in the malphigian tubule. The expression of EST transcripts of the BmST clustered genes on chromosome 27 was distributed in various tissues like testis, ovary, silk gland, malphigian tubule, maxillary galea, prothoracic gland, epidermis, fat body and midgut. Three sugar transporter genes (BmST) were constitutively expressed in the susceptible race and were down regulated upon BmNPV infection at 12h post infection (hpi). The expression pattern of these three genes was validated through real-time PCR in the midgut tissues at different time intervals from 0 to 30hpi. In the susceptible B. mori race, expression of sugar transporter genes was constitutively expressed making the host succumb to viral infection. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Chinese Calligraphy.

    ERIC Educational Resources Information Center

    Stanford Univ., CA. Stanford Program on International and Cross Cultural Education.

    This unit is designed to introduce secondary or post-secondary students to the ancient art of Chinese calligraphy through step-by-step instructions on writing Chinese characters. Because each character is made up of a series of single brush strokes, it is believed that if students learn to recognize these as components of completed characters, the…

  7. Chinese Cooking.

    ERIC Educational Resources Information Center

    Kane, Tony

    This unit, intended for secondary level students, is a general introduction to Chinese cooking. It is meant to inform students about the origins of Chinese cooking styles in their various regional manifestations, and it can be used to discuss how and why different cultures develop different styles of cooking. The first part of the unit, adapted…

  8. Chinese Cooking.

    ERIC Educational Resources Information Center

    Kane, Tony

    This unit, intended for secondary level students, is a general introduction to Chinese cooking. It is meant to inform students about the origins of Chinese cooking styles in their various regional manifestations, and it can be used to discuss how and why different cultures develop different styles of cooking. The first part of the unit, adapted…

  9. Combined Effect of Cameo2 and CBP on the Cellular Uptake of Lutein in the Silkworm, Bombyx mori

    PubMed Central

    Dong, Xiao-Long; Chai, Chun-Li; Pan, Cai-Xia; Tang, Hui; Chen, Yan-Hong; Dai, Fang-Yin; Pan, Min-Hui; Lu, Cheng

    2014-01-01

    Formation of yellow-red color cocoons in the silkworm, Bombyx mori, occurs as the result of the selective delivery of carotenoids from the midgut to the silk gland via the hemolymph. This process of pigment transport is thought to be mediated by specific cellular carotenoids carrier proteins. Previous studies indicated that two proteins, Cameo2 and CBP, are associated with the selective transport of lutein from the midgut into the silk gland in Bombyx mori. However, the exact roles of Cameo2 and CBP during the uptake and transport of carotenoids are still unknown. In this study, we investigated the respective contributions of these two proteins to lutein and β-carotene transport in Bombyx mori as well as commercial cell-line. We found that tissues, expressed both Cameo2 and CBP, accumulate lutein. Cells, co-expressed Cameo2 and CBP, absorb 2 fold more lutein (P<0.01) than any other transfected cells, and the rate of cellular uptake of lutein was concentration-dependent and reached saturation. From immunofluorescence staining, confocal microscopy observation and western blot analysis, Cameo2 was localized at the membrane and CBP was expressed in the cytosol. What’s more, bimolecular fluorescence complementation analysis showed that these two proteins directly interacted at cellular level. Therefore, Cameo2 and CBP are necessarily expressed in midguts and silk glands for lutein uptake in Bombyx mori. Cameo2 and CBP, as the membrane protein and the cytosol protein, respectively, have the combined effect to facilitate the cellular uptake of lutein. PMID:24475153

  10. Heterosis and differential gene expression in hybrids and parents in Bombyx mori by digital gene expression profiling

    PubMed Central

    Wang, Hua; Fang, Yan; Wang, Lipeng; Zhu, Wenjuan; Ji, Haipeng; Wang, Haiying; Xu, Shiqing; Sima, Yanghu

    2015-01-01

    Heterosis is a concern to all breeders, but the mechanism of heterosis remains unknown. In F1 organisms, genetic material is inherited from the two parents and theoretically, heterosis might be caused by differences in gene expression or modification. Differential gene expression was analyzed in hybrids and parents in Bombyx mori. The results showed that there were significant changes in gene expression in the fat body involving biological regulation, cellular and metabolic processes. Consistent trends in expression patterns covering different hybrid combinations were seen in 74 genes. Moreover, these differential gene expression patterns included overdominance, dominance, and additive effects. By correlating these patterns with economic traits, a potential relationship was found. Differential gene expression was seen in different cross combinations and in different sexes. In addition, a regulatory mechanism involving metabolism and ErbB signaling pathways was also found, suggesting that such a network might also be related to heterosis in Bombyx mori. Together, our data provide a comprehensive overview and useful resource for transcriptional analysis of heterosis of Bombyx mori. PMID:25736158

  11. Heterosis and differential gene expression in hybrids and parents in Bombyx mori by digital gene expression profiling.

    PubMed

    Wang, Hua; Fang, Yan; Wang, Lipeng; Zhu, Wenjuan; Ji, Haipeng; Wang, Haiying; Xu, Shiqing; Sima, Yanghu

    2015-03-04

    Heterosis is a concern to all breeders, but the mechanism of heterosis remains unknown. In F1 organisms, genetic material is inherited from the two parents and theoretically, heterosis might be caused by differences in gene expression or modification. Differential gene expression was analyzed in hybrids and parents in Bombyx mori. The results showed that there were significant changes in gene expression in the fat body involving biological regulation, cellular and metabolic processes. Consistent trends in expression patterns covering different hybrid combinations were seen in 74 genes. Moreover, these differential gene expression patterns included overdominance, dominance, and additive effects. By correlating these patterns with economic traits, a potential relationship was found. Differential gene expression was seen in different cross combinations and in different sexes. In addition, a regulatory mechanism involving metabolism and ErbB signaling pathways was also found, suggesting that such a network might also be related to heterosis in Bombyx mori. Together, our data provide a comprehensive overview and useful resource for transcriptional analysis of heterosis of Bombyx mori.

  12. Modulation of gene expression by nutritional state and hormones in Bombyx larvae in relation to its growth period.

    PubMed

    Thounaojam, Bembem; Keshan, Bela

    2017-08-23

    Insect growth and development are mainly regulated via synchronization of many extrinsic and intrinsic factors such as nutrition and hormones. Previously we have demonstrated that larval growth period influences the effect of insulin on the accumulation of glycogen in the fat body of Bombyx larvae. In the present study we demonstrate that Bombyx larvae at the terminal growth period (TGP, after critical weight) had a significantly greater increase in the expression level of Akt in the fat body than at the active growth period (AGP, before critical weight). The larvae at TGP also showed an increase in the expression level of ecdysone receptors (EcRB1 and USP1) and ecdysone-induced early genes (E75A and broad). The treatment of bovine insulin and methoprene to larvae at AGP induced the transcript levels of Akt, irrespective of the nutritional status of the larvae. However, in larvae at TGP, insulin repressed the transcript level of Akt. On contrary, 20-hydroxyecdysone induced the expression level of Akt in TGP larvae, but at feeding only. Insulin and 20E thus showed an antagonistic action on the Akt expression level in TGP larvae under feeding. The studies thus showed that larval growth period influences the expression level of Akt and ecdysone receptors in Bombyx. Further, the growth period and nutrition modulate the effect of exogenous hormones on Akt expression. Copyright © 2017. Published by Elsevier B.V.

  13. Disruption of diapause induction by TALEN-based gene mutagenesis in relation to a unique neuropeptide signaling pathway in Bombyx

    PubMed Central

    Shiomi, Kunihiro; Takasu, Yoko; Kunii, Masayo; Tsuchiya, Ryoma; Mukaida, Moeka; Kobayashi, Masakazu; Sezutsu, Hideki; Ichida Takahama, Masatoshi; Mizoguchi, Akira

    2015-01-01

    The insect neuropeptide family FXPRLa, which carries the Phe-Xaa-Pro-Arg-Leu-NH2 sequence at the C-terminus, is involved in many physiological processes. Although ligand–receptor interactions in FXPRLa signaling have been examined using in vitro assays, the correlation between these interactions and in vivo physiological function is unclear. Diapause in the silkworm, Bombyx mori, is thought to be elicited by diapause hormone (DH, an FXPRLa) signaling, which consists of interactions between DH and DH receptor (DHR). Here, we performed transcription activator-like effector nuclease (TALEN)-based mutagenesis of the Bombyx DH-PBAN and DHR genes and isolated the null mutants of these genes in a bivoltine strain. All mutant silkworms were fully viable and showed no abnormalities in the developmental timing of ecdysis or metamorphosis. However, female adults oviposited non-diapause eggs despite diapause-inducing temperature and photoperiod conditions. Therefore, we conclude that DH signaling is essential for diapause induction and consists of highly sensitive and specific interactions between DH and DHR selected during ligand–receptor coevolution in Bombyx mori. PMID:26497859

  14. Baculovirus LEF-11 Hijack Host ATPase ATAD3A to Promote Virus Multiplication in Bombyx mori cells

    PubMed Central

    Dong, Zhan-Qi; Hu, Nan; Dong, Fei-Fan; Chen, Ting-Ting; Jiang, Ya-Ming; Chen, Peng; Lu, Cheng; Pan, Min-Hui

    2017-01-01

    Research on molecular mechanisms that viruses use to regulate the host apparatus is important in virus infection control and antiviral therapy exploration. Our previous research showed that the Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 localized to dense regions of the cell nucleus and is required for viral DNA replication. Herein, we examined the mechanism of LEF-11 on BmNPV multiplication and demonstrated that baculovirus LEF-11 interacts with Bombyx mori ATAD3A and HSPD1 (HSP60) protein. Furthermore, we showed that LEF-11 has the ability to induce and up-regulate the expression of ATAD3A and HSPD1, phenomena that were both reversed upon knockdown of lef-11. Our findings showed that ATAD3A and HSPD1 were necessary and contributed to BmNPV multiplication in Bombyx mori cells. Moreover, ATAD3A was found to directly interact with HSPD1. Interestingly, ATAD3A was required for the expression of HSPD1, while the knockdown of HSPD1 had no obvious effect on the expression level of ATAD3A. Taken together, the data presented in the current study demonstrated that baculovirus LEF-11 hijacks the host ATPase family members, ATAD3A and HSPD1, efficiently promote the multiplication of the virus. This study furthers our understanding of how baculovirus modulates energy metabolism of the host and provides a new insight into the molecular mechanisms of antiviral research. PMID:28393927

  15. Direct and indirect effects of RNA interference against pyridoxal kinase and pyridoxine 5'-phosphate oxidase genes in Bombyx mori.

    PubMed

    Huang, ShuoHao; Yao, LiLi; Zhang, JianYun; Huang, LongQuan

    2016-08-01

    Vitamin B6 comprises six interconvertible pyridine compounds (vitamers), among which pyridoxal 5'-phosphate is a coenzyme involved in a high diversity of biochemical reactions. Humans and animals obtain B6 vitamers from diet, and synthesize pyridoxal 5'-phosphate by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. Currently, little is known on how pyridoxal 5'-phosphate biosynthesis is regulated, and pyridoxal 5'-phosphate is supplied to meet their requirement in terms of cofactor. Bombyx mori is a large silk-secreting insect, in which protein metabolism is most active, and the vitamin B6 demand is high. In this study, we successfully down-regulated the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase by body cavity injection of synthesized double-stranded small interfering RNA to 5th instar larvae of Bombyx mori, and analyzed the gene transcription levels of pyridoxal 5'-phosphate dependent enzymes, phosphoserine aminotransferase and glutamic-oxaloacetic transaminase. Results show that the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase has a greater impact on the gene transcription of enzymes using pyridoxal 5'-phosphate as a cofactor in Bombyx mori. Our study suggests that pyridoxal 5'-phosphate biosynthesis and dynamic balance may be regulated by genetic networks. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Precise genome editing in the silkworm Bombyx mori using TALENs and ds- and ssDNA donors - A practical approach.

    PubMed

    Takasu, Yoko; Kobayashi, Isao; Tamura, Toshiki; Uchino, Keiro; Sezutsu, Hideki; Zurovec, Michal

    2016-11-01

    Engineered nucleases are able to introduce double stranded breaks at desired genomic locations. The breaks can be repaired by an error-prone non-homologous end joining (NHEJ) mechanism, or the repair process can be exploited to introduce precise DNA modifications by homology-directed repair (HDR) when provided with a suitable donor template. We designed a series of DNA donors including long dsDNA plasmids as well as short ssDNA oligonucleotides and compared the effectiveness of their utilization during gene targeting with highly efficient transcription activator-like effector nucleases (TALENs). While the use of long dsDNA donors for the incorporation of larger DNA fragments in Bombyx is still a problem, short single-stranded oligodeoxynucleotides (ssODNs) are incorporated quite efficiently. We show that appropriately designed ssODNs were integrated into germ cells in up to 79% of microinjected individuals and describe in more detail the conditions for the precise genome editing of Bombyx genes. We specify the donor sequence requirements that affected knock-in efficiency, and demonstrate the successful applications of this method of sequence deletion, insertion and replacement in the Bombyx genome. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Defense role of the cocoon in the silk worm Bombyx mori L.

    PubMed

    Pandiarajan, Jeyaraj; Cathrin, Britto P; Pratheep, Thangaraj; Krishnan, Muthukalingan

    2011-11-15

    Silk from the domesticated silk worm Bombyx mori procures foreign body response naturally, so it has been utilized as a biomaterial for decades. In India the prime focus of the sericulture industry is to improve silk production with high quality silk. Naturally, the silk worm builds its cocoon not only with silk proteins, but also with antimicrobial proteins to avoid infection since the cocoon is non-motile and non-feeding. The aim of the present study is to elucidate the antimicrobial proteins that persist in the cocoon of the silk worm Bombyx mori. At the pupal stage, the silk worm cocoon shell extract was prepared from the day of pupation (P0) to the day of natural rupture of the cocoon for the eclosion of moth (NR). Using the cocoon shell extract a microbial susceptibility test was performed by the disc diffusion method against the microbes Escherchia coli, Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The development of a zone of inhibition against the microbes confirmed the presence of antimicrobial/immunogenic activity of the cocoon shell extract. For further analysis, the cocoon shell extract was subjected to 7-15% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The protein profile of the cocoon extract revealed the coomassie blue stained bands resolved from the 150-15 kDa molecular range. Interestingly, a polypeptide localized at around 29 kDa showed remarkable expressional changes during the development of pupa. To characterize the 29 kDa protein, it was eluted from the gel, digested with trypsin and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The trypsin-digested peptide peaks were analyzed through MASCOT and peptides were matched with the NCBI nr database. The peptides were very well matched with the 18 wheeler protein, which is reported to be responsible for innate immunity, belonging to the Toll family in insects and responsible for cellular

  18. Development stage-specific expression of fibroin in the silk worm Bombyx mori is regulated translationally.

    PubMed

    Patel, C V; Gopinathan, K P

    1991-01-01

    The contents of fibroin H RNA as a function of development have been quantitated in the posterior silk glands of Bombyx mori larvae on different days of 4th and 5th instars. The fibroin RNA levels increased during the feeding stages of larvae and the RNA got completely degraded during the interim moult. The patterns of accumulation of fibroin RNA were similar in both the instars. Although there was considerable increase in the fibroin RNA content during the 5th larval instar, the relative abundance of fibroin RNA in the total RNA was fairly constant during the 4th and 5th instars. The increased content of fibroin RNA in 5th instar was the consequence of an overall increase in transcription accompanying the development progress, rather than specific increase only in fibroin transcription. The contents of fibroin protein in the 4th and 5th instars of development have also been quantitated making use of a sensitive radioimmune assay with a purified, antifibroin antibody. There were substantial differences between 4th and 5th instars in the absolute fibroin contents as well as the relative proportion of fibroin in the total proteins. These results implied that although the fibroin gene was transcribed at the same efficiency during the 4th and 5th instars, the translational efficiency was much lower during the 4th instar. The extent of polyadenylation of fibroin RNA was similar in both instars. However, there was a two-fold increase in the polysome association of fibroin RNA in the 5th instar. Over and above this, there was substantial increase during the 5th instar in the contents of those tRNAs. (e.g. Gly, Ala and Ser) which are abundantly represented in fibroin and therefore directly related to the expression of fibroin. The increased polysome association of fibroin mRNA and the adequate supply of cognate tRNAs in the 5th instar, together contributes to the translational regulation of fibroin in a developmental stage-specific manner. Based on these observations, we

  19. Changing Chinese

    ERIC Educational Resources Information Center

    Sung, Betty Lee

    1977-01-01

    Notes that in many ways the makeup of today's Chinese immigrants tends toward the extremes. At one end they are highly educated, at the other, they are the beneficiaries of the nonquota provisions of the immigration law. (Author/AM)

  20. Analysis of the Bombyx mori nucleopolyhedrovirus ie-1 promoter in insect, mammalian, plant, and bacterial cells.

    PubMed

    Fujita, Ryosuke; Ono, Chikako; Ono, Isamu; Asano, Shin-Ichiro; Bando, Hisanori

    2015-09-04

    The Bombyx mori nucleopolyhedrovirus (BmNPV) ie-1 promoter exhibits strong transcriptional activity and is used in transient foreign gene expression systems in insect cells. In a reporter assay experiment using the BmNPV ie-1 promoter, we found that it exhibited activity even in non-host mammalian BHK cells, plant BY-2 cells, and also bacterial Escherichia coli cells. An analysis using a deletion series of the BmNPV ie-1 promoter demonstrated that the core promoter region of this promoter was sufficient to display promoter activity in BHK cells, BY-2 cells, and E. coli cells, whereas upstream elements were required for higher activity in insect cells. Furthermore, we found that the BmNPV ie-1 promoter exhibited sufficient activity for a β-galactosidase assay in E. coli cells. The results obtained here suggest that the BmNPV ie-1 promoter has potential as a universal promoter for transient expression systems in insect, mammalian, plant, and bacterial cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Regulation of gene expression in prediapausing embryos of the silkworm, Bombyx mori: pattern of protein synthesis.

    PubMed

    Dorel, C; Coulon, M

    1988-03-01

    Specific qualitative and quantitative changes in protein synthesis occur from the fertilization to the onset of diapause in the silkworm. We have used two-dimensional gel electrophoresis to analyse the patterns of proteins synthesized in prediapausing eggs of Bombyx. This analysis has been carried out with in vivo labelled polypeptides and with proteins synthesized in vitro by RNA isolated at different stages. The oocyte contains an abundant supply of diverse mRNA which are translatable in vitro. A set of proteins with molecular weight range of 68,000 to 74,000 and isoelectric points of 5.85-5.95 (hereafter referred to as No. 30) is specific of the germ-anlage stage. Transcripts encoding the No. 30 proteins are not detectable in oocytes, and inhibition of transcription by actinomycin D indicates that No. 30 mRNA are synthesized de novo. Treating eggs at the germ-anlage stage with 4 N HCl at 46 degrees C prevents diapause and is accompanied by overproduction of No. 30 protein. The induction of No. 30 synthesis is also the main event of the heat shock response. The implications of these findings in relation to early embryonic development and prevention of diapause are discussed.

  2. SilkDB v2.0: a platform for silkworm (Bombyx mori ) genome biology.

    PubMed

    Duan, Jun; Li, Ruiqiang; Cheng, Daojun; Fan, Wei; Zha, Xingfu; Cheng, Tingcai; Wu, Yuqian; Wang, Jun; Mita, Kazuei; Xiang, Zhonghuai; Xia, Qingyou

    2010-01-01

    The SilkDB is an open-access database for genome biology of the silkworm (Bombyx mori). Since the draft sequence was completed and the SilkDB was first released 5 years ago, we have collaborated with other groups to make much remarkable progress on silkworm genome research, such as the completion of a new high-quality assembly of the silkworm genome sequence as well as the construction of a genome-wide microarray to survey gene expression profiles. To accommodate these new genomic data and house more comprehensive genomic information, we have reconstructed SilkDB database with new web interfaces. In the new version (v2.0) of SilkDB, we updated the genomic data, including genome assembly, gene annotation, chromosomal mapping, orthologous relationship and experiment data, such as microarray expression data, Expressed Sequence Tags (ESTs) and corresponding references. Several new tools, including SilkMap, Silkworm Chromosome Browser (SCB) and BmArray, are developed to access silkworm genomic data conveniently. SilkDB is publicly available at the new URL of http://www.silkdb.org.

  3. FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori.

    PubMed

    Long, Ding-Pei; Zhao, Ai-Chun; Chen, Xue-Jiao; Zhang, Yang; Lu, Wei-Jian; Guo, Qing; Handler, Alfred M; Xiang, Zhong-Huai

    2012-01-01

    A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.

  4. Identification and characterization of Polycomb group genes in the silkworm, Bombyx mori.

    PubMed

    Li, Zhiqing; Tatsuke, Tsuneyuki; Sakashita, Kosuke; Zhu, Li; Xu, Jian; Mon, Hiroaki; Lee, Jae Man; Kusakabe, Takahiro

    2012-05-01

    Polycomb group (PcG) proteins are involved in chromatin modifications for maintaining gene repression that play important roles in the regulation of gene expression, tumorigenesis, chromosome X-inactivation, and genomic imprinting in Drosophila melanogaster, mammals, and even plants. To characterize the orthologs of PcG genes in the silkworm, Bombyx mori, 13 candidates were identified from the updated silkworm genome sequence by using the fruit fly PcG genes as queries. Comparison of the silkworm PcG proteins with those from other insect species revealed that the insect PcG proteins shared high sequence similarity. High-level expressions of all the silkworm PcG genes were maintained through day 2 to day 7 of embryogenesis, and tissue microarray data on day 3 of the fifth instar larvae showed that their expression levels were relatively low in somatic tissues, except for Enhancer of zeste (E(Z)). In addition, knockdown of each PRC2 component, such as E(Z), Extra sex combs (ESC), and Suppressor of zeste 12 (SU(Z)12), considerably decreased the global levels of H3K27me3 but not of H3K27me2. Taken together, these results suggest that insect PcG proteins are highly conserved during evolution and might play similar roles in embryogenesis.

  5. Identification, genomic organization and expression pattern of glutathione S-transferase in the silkworm, Bombyx mori.

    PubMed

    Yu, Quanyou; Lu, Cheng; Li, Bin; Fang, Shoumin; Zuo, Weidong; Dai, Fangyin; Zhang, Ze; Xiang, Zhonghuai

    2008-12-01

    Glutathione S-transferases (GSTs) are a multifunctional supergene family and some play an important role in insecticide resistance. We have identified 23 putative cytosolic GSTs by searching the new assembly of the Bombyx mori genome sequence. Phylogenetic analyses on the amino acid sequences reveal that 21 of the B. mori GSTs fall into six classes represented in other insects, the other two being unclassified. The majority of the silkworm GSTs belong to the Delta, Epsilon, and Omega classes. Most members of each class are tandemly arranged in the genome, except for the Epsilon GSTs. Expressed sequence tags (ESTs) corresponding to 19 of the 23 GSTs were found in available databases. Furthermore RT-PCR experiments detected expression of all the GSTs in multiple tissues on day 3 of fifth instar larvae. Surprisingly, we found little or no expression of most Delta and Epsilon GSTs in the fat body, which is thought to be the main detoxification organ. This may explain the sensitivity of the silkworm to certain insecticides. Our data provide some insights into the evolution of the B. mori GST family and the functions of individual GST enzymes.

  6. A discovery of novel microRNAs in the silkworm (Bombyx mori) genome.

    PubMed

    Yu, Xiaomin; Zhou, Qing; Cai, Yimei; Luo, Qibin; Lin, Hongbin; Hu, Songnian; Yu, Jun

    2009-12-01

    MicroRNAs (miRNAs) are pivotal regulators involved in various physiological and pathological processes via their post-transcriptional regulation of gene expressions. We sequenced 14 libraries of small RNAs constructed from samples spanning the life cycle of silkworms, and discovered 50 novel miRNAs previously not known in animals and verified 43 of them using stem-loop RT-PCR. Our genome-wide analyses of 27 species-specific miRNAs suggest they arise from transposable elements, protein-coding genes duplication/transposition and random foldback sequences; which is consistent with the idea that novel animal miRNAs may evolve from incomplete self-complementary transcripts and become fixed in the process of co-adaptation with their targets. Computational prediction suggests that the silkworm-specific miRNAs may have a preference of regulating genes that are related to life-cycle-associated traits, and these genes can serve as potential targets for subsequent studies of the modulating networks in the development of Bombyx mori.

  7. Functional characterization of the vitellogenin promoter in the silkworm, Bombyx mori.

    PubMed

    Xu, J; Wang, Y Q; Li, Z Q; Ling, L; Zeng, B S; You, L; Chen, Y Z; Aslam, A F M; Huang, Y P; Tan, A J

    2014-10-01

    Genetic transformation and genome editing technologies have been successfully established in the lepidopteran insect model, the domesticated silkworm, Bombyx mori, providing great potential for functional genomics and practical applications. However, the current lack of cis-regulatory elements in B. mori gene manipulation research limits further exploitation in functional gene analysis. In the present study, we characterized a B. mori endogenous promoter, Bmvgp, which is a 798-bp DNA sequence adjacent to the 5'-end of the vitellogenin gene (Bmvg). PiggyBac-based transgenic analysis shows that Bmvgp precisely directs expression of a reporter gene, enhanced green fluorescent protein (EGFP), in a sex-, tissue- and stage-specific manner. In transgenic animals, EGFP expression can be detected in the female fat body from larval-pupal ecdysis to the following pupal and adult stage. Furthermore, in vitro and in vivo experiments revealed that EGFP expression can be activated by 20-hydroxyecdysone, which is consistent with endogenous Bmvg expression. These data indicate that Bmvgp is an effective endogenous cis-regulatory element in B. mori.

  8. Role of GTP-CHI links PAH and TH in melanin synthesis in silkworm, Bombyx mori.

    PubMed

    Chen, Ping; Wang, Jiying; Li, Haiyin; Li, Yan; Chen, Peng; Li, Tian; Chen, Xi; Xiao, Junjie; Zhang, Liang

    2015-08-10

    In insects, pigment patterns are formed by melanin, ommochromes, and pteridines. Here, the effects of pteridine synthesis on melanin formation were studied using 4th instar larvae of a wild-type silkworm strain, dazao (Bombyx mori), with normal color and markings. Results from injected larvae and in vitro integument culture indicated that decreased activity of guanosine triphosphate cyclohydrolase I (GTP-CH I, a rate-limiting enzyme for pteridine synthesis), lowers BH4 (6R-l-erythro-5,6,7,8-tetrahydrobiopterin, a production correlated with GTP-CH I activity) levels and eliminates markings and coloration. The conversion of phenylalanine and tyrosine to melanin was prevented when GTP-CH I was inhibited. When BH4 was added, phenylalanine was converted to tyrosine, and the tyrosine concentration increased. Tyrosine was then converted to melanin to create normal markings and coloration. Decreasing GTP-CH I activity did not affect L-DOPA (3,4-l-dihydroxyphenylalanine). GTP-CH I affected melanin synthesis by generating the BH4 used in two key reaction steps: (1) conversion of phenylalanine to tyrosine by PAH (phenylalanine hydroxylase) and (2) conversion of tyrosine to L-DOPA by TH (tyrosine hydroxylase). Expression profiles of BmGTPCH Ia, BmGTPCH Ib, BmTH, and BmPAH in the integument were consistent with the current findings.

  9. Combinatory annotation of cell membrane receptors and signalling pathways of Bombyx mori prothoracic glands.

    PubMed

    Moulos, Panagiotis; Samiotaki, Martina; Panayotou, George; Dedos, Skarlatos G

    2016-08-30

    The cells of prothoracic glands (PG) are the main site of synthesis and secretion of ecdysteroids, the biochemical products of cholesterol conversion to steroids that shape the morphogenic development of insects. Despite the availability of genome sequences from several insect species and the extensive knowledge of certain signalling pathways that underpin ecdysteroidogenesis, the spectrum of signalling molecules and ecdysteroidogenic cascades is still not fully comprehensive. To fill this gap and obtain the complete list of cell membrane receptors expressed in PG cells, we used combinatory bioinformatic, proteomic and transcriptomic analysis and quantitative PCR to annotate and determine the expression profiles of genes identified as putative cell membrane receptors of the model insect species, Bombyx mori, and subsequently enrich the repertoire of signalling pathways that are present in its PG cells. The genome annotation dataset we report here highlights modules and pathways that may be directly involved in ecdysteroidogenesis and aims to disseminate data and assist other researchers in the discovery of the role of such receptors and their ligands.

  10. Impact of processing parameters on the haemocompatibility of Bombyx mori silk films

    PubMed Central

    Seib, F. Philipp; Maitz, Manfred F.; Hu, Xiao; Werner, Carsten; Kaplan, David L.

    2013-01-01

    Silk has traditionally been used for surgical sutures due to its lasting strength and durability; however, the use of purified silk proteins as a scaffold material for vascular tissue engineering goes beyond traditional use and requires application-orientated biocompatibility testing. For this study, a library of Bombyx mori silk films was generated and exposed to various solvents and treatment conditions to reflect current silk processing techniques. The films, along with clinically relevant reference materials, were exposed to human whole blood to determine silk blood compatibility. All substrates showed an initial inflammatory response comparable to polylactide-co-glycolide (PLGA), and a low to moderate haemostasis response similar to polytetrafluoroethylene (PTFE) substrates. In particular, samples that were water annealed at 25 °C for 6 h demonstrated the best blood compatibility based on haemostasis parameters (e.g. platelet decay, thrombin-antithrombin complex, platelet factor 4, granulocytes-platelet conjugates) and inflammatory parameters (e.g. C3b, C5a, CD11b, surface-associated leukocytes). Multiple factors such as treatment temperature and solvent influenced the biological response, though no single physical parameter such as β-sheet content, isoelectric point or contact angle accurately predicted blood compatibility. These findings, when combined with prior in vivo data on silk, support a viable future for silk-based vascular grafts. PMID:22079005

  11. Immune signaling pathways activated in response to different pathogenic micro-organisms in Bombyx mori.

    PubMed

    Liu, Wei; Liu, Jiabin; Lu, Yahong; Gong, Yongchang; Zhu, Min; Chen, Fei; Liang, Zi; Zhu, Liyuan; Kuang, Sulan; Hu, Xiaolong; Cao, Guangli; Xue, Renyu; Gong, Chengliang

    2015-06-01

    The JAK/STAT, Toll, Imd, and RNAi pathways are the major signaling pathways associated with insect innate immunity. To explore the different immune signaling pathways triggered in response to pathogenic micro-organism infections in the silkworm, Bombyx mori, the expression levels of the signal transducer and activator of transcription (BmSTAT), spatzle-1 (Bmspz-1), peptidoglycan-recognition protein LB (BmPGRP-LB), peptidoglycan-recognition protein LE (BmPGRP-LE), argonaute 2 (Bmago2), and dicer-2 (Bmdcr2) genes after challenge with Escherichia coli (E. coli), Serratiamarcescens (Sm), Bacillus bombyseptieus (Bab), Beauveriabassiana (Beb), nucleopolyhedrovirus (BmNPV), cypovirus (BmCPV), bidensovirus (BmBDV), or Nosemabombycis (Nb) were determined using real-time PCR. We found that the JAK/STAT pathway could be activated by challenge with BmNPV and BmBDV, the Toll pathway could be most robustly induced by challenge with Beb, the Imd pathway was mainly activated in response to infection by E. coli and Sm, and the RNAi pathway was not activated by viral infection, but could be triggered by some bacterial infections. These findings yield insights into the immune signaling pathways activated in response to different pathogenic micro-organisms in the silkworm.

  12. Processing and characterization of silk sericin from Bombyx mori and its application in biomaterials and biomedicines.

    PubMed

    Cao, Ting-Ting; Zhang, Yu-Qing

    2016-04-01

    Bombyx mori silk is composed of 60-80% fibroin, 15-35% sericin and 1-5% non-sericin component including wax, pigments, sugars and other impurities. For two decades, the protein-based silk fibroin was extensively used in the research and development of medical biomaterials and biomedicines. Sericin is frequently ignored and abandoned as a byproduct or waste in the processing of traditional silk fabrics, silk floss or modern silk biomaterials. However, similar to fibroin, sericin is not only a highly useful biological material, but also a lot of biological activity. Moreover, the non-sericin component present with sericin in the cocoon shell also has a strong biological activity. In this review, the extraction and recovery methods of sericin and the non-sericin component from the cocoon layer are reported, and their composition, properties and biological activity are described to produce a comprehensive report on biomedical materials and biological drugs. In addition, related problems or concerns present in the research and development of sericin are discussed, and a potential application of sericin in sustainable development is also presented. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Analysis of proteome dynamics inside the silk gland lumen of Bombyx mori

    PubMed Central

    Dong, Zhaoming; Zhao, Ping; Zhang, Yan; Song, Qianru; Zhang, Xiaolu; Guo, Pengchao; Wang, Dandan; Xia, Qingyou

    2016-01-01

    The silk gland is the only organ where silk proteins are synthesized and secreted in the silkworm, Bombyx mori. Silk proteins are stored in the lumen of the silk gland for around eight days during the fifth instar. Determining their dynamic changes is helpful for clarifying the secretion mechanism of silk proteins. Here, we identified the proteome in the silk gland lumen using liquid chromatography–tandem mass spectrometry, and demonstrated its changes during two key stages. From day 5 of the fifth instar to day 1 of wandering, the abundances of fibroins, sericins, seroins, and proteins of unknown functions increased significantly in different compartments of the silk gland lumen. As a result, these accumulated proteins constituted the major cocoon components. In contrast, the abundances of enzymes and extracellular matrix proteins decreased in the silk gland lumen, suggesting that they were not the structural constituents of silk. Twenty-five enzymes may be involved in the regulation of hormone metabolism for proper silk gland function. In addition, the metabolism of other non-proteinous components such as chitin and pigment were also discussed in this study. PMID:27102218

  14. Involvement of Bombyx mori nucleopolyhedrovirus ORF41 (Bm41) in BV production and ODV envelopment

    SciTech Connect

    Tian Caihong; Zhao Jinfang; Xu Yipeng; Xue Jian; Zhang Baoqin; Cui Yingjun; Zhang Minjuan; Bao Yanyuan; Zhang Chuanxi

    2009-04-25

    Bombyx mori nucleopolyhedrovirus (BmNPV) ORF41 (Bm41), homologous to Ac52, is a gene present in most lepidopteran nucleopolyhedroviruses. Bm41 transcripts and encoded protein in BmNPV-infected cells can be detected from 3 and 6 h post-infection, respectively. Immunoassays have shown that Bm41 is not a viral structural protein and is detected in both the nuclei and cytoplasm of infected cells. A Bm41-disrupted virus (vBm{sup De}) and a repaired virus (vBm{sup Re}) were generated to investigate the function of Bm41. The results showed that Bm41 was essential for viral replication, and the disruption of Bm41 resulted in a much lower viral titer. Transmission electron microscopy revealed that disruption of Bm41 affected normal nucleocapsid envelopment and polyhedra formation in the nucleus. The disruption of Bm41 might severely affect odv-ec27 and polyhedrin expression. The disrupted virus reduced BmNPV infectivity in an LD{sub 50} bioassay and took 18-23 h longer to kill larvae than wild-type virus in an LT{sub 50} bioassay.

  15. Fine Mapping of a Degenerated Abdominal Legs Mutant (Edl) in Silkworm, Bombyx mori

    PubMed Central

    Liu, Meijing; Hu, Hai; Li, Zhiquan; Xiang, Zhonghuai; Dai, Fangyin; Lu, Cheng

    2017-01-01

    In insects, abdominal appendages, also called prolegs, vary due to adaptive evolution. Mutations on prolegs within species provide insights to better understand the mechanisms underlying appendage development and diversity. In silkworm Bombyx mori, extra-crescents and degenerated abdominal legs (Edl) mutant, belonging to the E pseudoallele group, is a spontaneous mutation that adds crescents and degenerates prolegs on the third abdominal segment (A3). This mutation may be a homeotic transformation of A3 to A2. In this study, the Edl locus was mapped within approximately a 211 Kb region that is 10 Kb upstream of Bmabdominal-A (Bmabd-A). RT-quantitative PCR (RT-qPCR) and Western blot analysis of Bmabd-A expression showed a slight but significant decrease, while the expression of BmUltrabithorax (BmUbx) was up-regulated in the Edl mutant compared to wildtype (Dazao). Moreover, we also found that BmDistal-less (BmDll), which regulated the development of distal proleg structures, was missing at the tips of the A3 prolegs in the Edl mutant compared to BmDll expression in normally developed prolegs in both the wildtype and mutant. Collectively, we identified approximately a 211 Kb region in the Edl locus that regulates BmUbx and Bmabd-A expression and found that changes in BmUbx and Bmabd-A expression may lead to the loss of distal proleg structures in B. mori. PMID:28081147

  16. Nonvirus encoded proteins could be embedded into Bombyx mori cypovirus polyhedra.

    PubMed

    Zhang, Yi-Ling; Xue, Ren-Yu; Cao, Guang-Li; Meng, Xiang-Kun; Zhu, Yue-Xiong; Pan, Zhong-Hua; Gong, Cheng-Liang

    2014-01-01

    To explore whether the nonvirus encoded protein could be embedded into Bombyx mori cypovirus (BmCPV) polyhedra. The stable transformants of BmN cells expressing a polyhedrin (Polh) gene of BmCPV were constructed by transfection with a non-transposon derived vector containing a polh gene. The polyhedra were purified from the midguts of BmCPV-infected silkworms and the transformed BmN cells, respectively. The proteins embedded into polyhedra were determined by mass spectrometry analysis. Host derived proteins were detected in the purified polyhedra. Analysis of structure and hydrophilicity of embedded proteins indicated that the hydrophilic proteins, in structure, were similar to the left-handed structure of polyhedrin or the N-terminal domain of BmCPV structural protein VP3, which were easily embedded into the BmCPV polyhedra. The lysate of polyhedra purified from the infected transformation of BmN cells with modified B. mori baculovirus BmPAK6 could infect BmN cells, indicating that B. mori baculovirus could be embedded into BmCPV polyhedra. Both the purified polyhedra and its lysate could be coloured by X-gal, indicating that the β-galactosidase expressed by BmPAK6 could be incorporated into BmCPV polyhedra. These results suggested that some heterologous proteins and baculovirus could be embedded into polyhedra in an unknown manner.

  17. Thermal behavior of Bombyx mori silk: evolution of crystalline parameters, molecular structure, and mechanical properties.

    PubMed

    Martel, A; Burghammer, M; Davies, R J; Riekel, C

    2007-11-01

    The thermal behavior up to degradation of Bombyx mori silk has been studied by scanning synchrotron radiation microdiffraction, gel electrophoresis, and mechanical testing. The diffraction patterns from single baves can be separated into scattering from anisotropic crystalline beta-sheet domains and random short-range order. In contrast to dragline silk, scattering from oriented, short-range-order fibroin is not observed. The sheath of sericin proteins can be selectively probed by a microbeam and shows also principally random short-range-order domains with a small crystalline beta-sheet fraction. Microdiffraction experiments on single baves from 100 to 573 K show an increase in lattice expansion along the [010] chain-stacking direction above 200-250 K, which could be due to an increase in side-chain mobility. Degradation of the crystalline fraction commences at approximately 500 K, and the fibers have become amorphous at about 570 K with an onset of carbonization. Gel electrophoresis shows that the degradation of FibH molecules starts already at about 350 K, while FibL molecules start degrading at about 400 K. The mechanical properties of single baves such as strain-to-failure and tensile strength also start degrading at about 400 K, while the initial modulus increases up to about 475 K. It is proposed that this is due to the development of cross-linking in the short-range-order chain fraction.

  18. Detailed investigation of the sequential pathological changes in silkworm larvae infected with Bombyx densovirus type 1.

    PubMed

    Ito, Katsuhiko; Kidokoro, Kurako; Shimura, Sachiko; Katsuma, Susumu; Kadono-Okuda, Keiko

    2013-03-01

    Bombyx mori densovirus type 1 (BmDNV-1) is a pathogen causing flacherie disease in silkworms. BmDNV-1 multiplies only in the nuclei of the columnar cells of larval midgut epithelium. Although several immunohistochemical studies using anti-BmDNV-1 antibody have been reported to date, sequential pathological changes in BmDNV-1-infected larvae have not been completely elucidated. In this paper, sequential investigations were performed on the pathological features of BmDNV-1-infected larvae and BmDNV-1 propagation. Oral infection experiments using newly ecdysed 4th instar larvae revealed that the larvae began to die 9 days post infection (dpi), and the remaining died 10 dpi. Histological observations revealed phenotypic alterations in the midgut cells from 4 dpi, and complete disruption of the midgut structure at 9 dpi. Quantitative RT-PCR of two BmDNV-1 genes indicated that BmDNV-1 began to propagate from 4 dpi, and gradually increased until the larvae died. These expression patterns revealed marked correlation with the histological changes observed in the virus-infected midgut cells. Moreover, bioassays using larvae at various developmental stages clearly indicated that the pathogenicity of this virus is not dependent on the larval stage or the molting process.

  19. Novel Macula-Like Virus Identified in Bombyx mori Cultured Cells

    PubMed Central

    Katsuma, Susumu; Tanaka, Shinichiro; Omuro, Naoko; Takabuchi, Lisa; Daimon, Takaaki; Imanishi, Shigeo; Yamashita, Shuichi; Iwanaga, Masashi; Mita, Kazuei; Maeda, Susumu; Kobayashi, Masahiko; Shimada, Toru

    2005-01-01

    We identified a novel, 6,513-bp-long RNA, termed Bombyx mori macula-like latent virus (BmMLV) RNA, which abundantly expressed in B. mori cultured BmN cells. BmMLV RNA potentially encodes two proteins, putative RNA replicase and coat protein, which share structural features and sequence similarities with those of a plant RNA virus, the genus Maculavirus. Northern blot analysis showed that two transcripts were expressed in BmN cells: a 6.5-kb-long RNA, which contains both putative RNA replicase and coat protein genes, and a 1.2-kb-long RNA, which contains only a coat protein gene. Southern blot analysis showed that BmMLV RNA is not carried by the B. mori genome. RT-PCR analysis also revealed the presence of BmMLV RNA in several B. mori cell lines other than BmN cells, suggesting that BmMLV RNA latently exists in B. mori cultured cells. Infection studies showed that BmMLV virions were able to infect BmMLV-negative Spodoptera frugiperda Sf-9 cells and B. mori larvae. Electron microscopy and Northern blot analysis of a purified BmMLV revealed that isometric virions appear to be 28 to 30 nm in diameter and contain a 6.5-kb genomic RNA. These results showed that BmMLV is a novel macula-like virus infectious to and replicable in B. mori-derived cells. PMID:15827172

  20. Location of the Bombyx mori specificity domain on a Bacillus thuringiensis delta-endotoxin protein.

    PubMed Central

    Ge, A Z; Shivarova, N I; Dean, D H

    1989-01-01

    Bacillus thuringiensis produces different types of insecticidal crystal proteins (ICPs) or delta-endotoxins. In an effort to identify the insect specificity of ICP toxins, two icp genes were cloned into the Escherichia coli expression vector pKK223-3, and bioassays were performed with purified crystals. The type A protein [from an icpA1, or 4.5-kilobase (kb) gene, from B. thuringiensis var. kurstaki HD-1] was found to be 400 times more active against Bombyx mori than type C protein (from an icpC73, or 6.6-kb gene, from B. thuringiensis var. kurstaki HD-244). The type C protein was 9 times more active against Trichoplusia ni than the type A protein, while both have similar activity against Manduca sexta. To locate the specificity domain of the type A protein for B. mori, site-directed mutagenesis was used to introduce or remove restriction enzyme sites, facilitating the exchange of regions of the two genes. The hybrid genes were overexpressed, and purified ICP was used in bioassays. The B. mori specificity domain for the ICP A toxin is located in the amino-terminal portion of the hypervariable region between amino acids 332 and 450. PMID:2542961

  1. On the strength of β-sheet crystallites of Bombyx mori silk fibroin.

    PubMed

    Cheng, Yuan; Koh, Leng-Duei; Li, Dechang; Ji, Baohua; Han, Ming-Yong; Zhang, Yong-Wei

    2014-07-06

    Silk fibroin, a natural multi-domain protein, has attracted great attention due to its superior mechanical properties such as ultra-high strength and stretchability, biocompatibility, as well as its versatile biodegradability and processability. It is mainly composed of β-sheet crystallites and amorphous domains. Although its strength is well known to be controlled by the dissociation of protein chains from β-sheet crystallites, the way that water as the solvent affects its strength and the reason that its theoretically predicted strength is several times higher than experimental measurement remain unclear. We perform all-atom molecular dynamics simulations on a β-sheet crystallite of Bombyx mori silk. We find that water solvent reduces the number and strength of hydrogen bonds between β-chains, and thus greatly weakens the strength of silk fibroin. By dissociating protein chains at different locations from the crystallite, we also find that the pulling strength for the interior chains is several times higher than that for the surface/corner chains, with the former being consistent with the theoretically predicted value, while the latter on par with the experimental value. It is shown that the weakest rupture strength controls the failure strength of silk fibre. Hence, this work sheds light on the role of water in the strength of silk fibroin and also provides clues on the origin of the strength difference between theory and experiment.

  2. Identification and characterization of three novel hemocyte-specific promoters in silkworm Bombyx mori.

    PubMed

    Zhang, Kui; Yu, Shuang; Su, Jingjing; Xu, Man; Tan, Peng; Zhang, Yajun; Xiang, Zhonghuai; Cui, Hongjuan

    2015-05-22

    Insect hemocytes play essential roles in the metabolism, metamorphosis and immunity, which are closely related events of growth and development. Here, four novel hemocyte-specific genes were obtained and conformed in our study, namely, Bmintβ2, Bmintβ3, BmCatO, and BmSw04862, respectively. Subsequently, their promoter sequences were cloned, and their activity in hemocytes, fat body, and silk gland were analyzed using recombinant AcNPV vector system in vivo. Our results showed that Bmintβ2, Bmintβ3, and BmCatO were hemocyte-specific promoters in the silkworm, Bombyx mori. Interestingly, Bmintβ2, and Bmintβ3 promoter regions were both located in their first intron. Further analysis of a series of BmCatO promoter truncations showed that a 254 bp region could function as a promoter element in the tissue-specificity expression. In summary, the results of this study revealed that we have identified three hemocyte-specific promoters in silkworm that will not only great significance for better understanding of hemocyte-specific gene, but also has potential applications in insect hematopoiesis and innate immunity research.

  3. Thiol peroxiredoxin, a novel allergen from Bombyx mori, modulates functions of macrophages and dendritic cells

    PubMed Central

    Wang, Hui; Hu, Wei; Liang, Zhiling; zeng, Lu; Li, Jianjie; Yan, Hao; Yang, Pingchang; Liu, Zhigang; Wang, Lianglu

    2016-01-01

    Bombyx mori (B.mori, also known as silkworm) plays a role in the pathogenesis of allergic diseases. However, its allergens are to be characterized. The aim of this paper is to identify new silkworm allergens. Two-dimensional gel electrophoresis (2-DE) and mass spectrometry were employed to separate and identify potential allergens from silkworm pupa. Six potential allergens were identified in this study. Thiol peroxiredoxin (TP), one of the 6 allergens, reacted to serum IgE from patients sensitized to silkworm. By sensitizing with TP allergic asthma like symptoms were induced in mice, including elevation of the levels of serum IgE, IL-4 from bronchoalveolar lavage fluid and culture supernatant of spleen cells. In vitro experiments showed that TP significantly induced RAW264.7 cells (a macrophage cell line) apoptosis via modulating the BCL2 and Caspase9 pathways. The levels of CD80, CD40, CD83 and TNF-α in DC2.4 cells (a dendritic cell line) were increased in the culture after exposure to TP. In summary, TP is an allergic component of silkworm. It induces allergic asthma, and modulates the functions of macrophages and dendritic cells. PMID:28078005

  4. Advanced technologies for genetically manipulating the silkworm Bombyx mori, a model Lepidopteran insect

    PubMed Central

    Xu, Hanfu; O'Brochta, David A.

    2015-01-01

    Genetic technologies based on transposon-mediated transgenesis along with several recently developed genome-editing technologies have become the preferred methods of choice for genetically manipulating many organisms. The silkworm, Bombyx mori, is a Lepidopteran insect of great economic importance because of its use in silk production and because it is a valuable model insect that has greatly enhanced our understanding of the biology of insects, including many agricultural pests. In the past 10 years, great advances have been achieved in the development of genetic technologies in B. mori, including transposon-based technologies that rely on piggyBac-mediated transgenesis and genome-editing technologies that rely on protein- or RNA-guided modification of chromosomes. The successful development and application of these technologies has not only facilitated a better understanding of B. mori and its use as a silk production system, but also provided valuable experiences that have contributed to the development of similar technologies in non-model insects. This review summarizes the technologies currently available for use in B. mori, their application to the study of gene function and their use in genetically modifying B. mori for biotechnology applications. The challenges, solutions and future prospects associated with the development and application of genetic technologies in B. mori are also discussed. PMID:26108630

  5. Effects of transient high temperature treatment on the intestinal flora of the silkworm Bombyx mori.

    PubMed

    Sun, Zhenli; Kumar, Dhiraj; Cao, Guangli; Zhu, Liyuan; Liu, Bo; Zhu, Min; Liang, Zi; Kuang, Sulan; Chen, Fei; Feng, Yongjie; Hu, Xiaolong; Xue, Renyu; Gong, Chengliang

    2017-06-13

    The silkworm Bombyx mori is a poikilotherm and is therefore sensitive to various climatic conditions. The influence of temperature on the intestinal flora and the relationship between the intestinal flora and gene expression in the silkworm remain unknown. In the present study, changes of the intestinal flora at 48, 96 and 144 h following transient high temperature treatment (THTT) of 37 °C for 8 h were investigated. According to principal component analysis, the abundances of Enterococcus and Staphylococcus showed a negative correlation with other dominant genera. After THTT, the gene expression levels of spatzle-1 and dicer-2 were increased and decreased, respectively, which suggested that the Toll and RNAi pathways were activated and suppressed, respectively. The species-gene expression matrix confirmed that the spatzle-1 and dicer-2 gene expression levels were negatively and positively correlated, respectively, with the abundance of Enterococcus and Staphylococcus in the control. The abundance of Variovorax post-THTT was positively correlated with the spatzle-1 gene expression level, whereas the community richness of Enterococcus was negatively correlated with the spatzle-1 gene expression level and positively correlated with the dicer-2. The results of the present investigation provide new evidence for understanding the relationships among THTT, intestinal flora and host gene expression.

  6. An integrated CRISPR Bombyx mori genome editing system with improved efficiency and expanded target sites.

    PubMed

    Ma, Sanyuan; Liu, Yue; Liu, Yuanyuan; Chang, Jiasong; Zhang, Tong; Wang, Xiaogang; Shi, Run; Lu, Wei; Xia, Xiaojuan; Zhao, Ping; Xia, Qingyou

    2017-02-09

    Genome editing enabled unprecedented new opportunities for targeted genomic engineering of a wide variety of organisms ranging from microbes, plants, animals and even human embryos. The serial establishing and rapid applications of genome editing tools significantly accelerated Bombyx mori (B. mori) research during the past years. However, the only CRISPR system in B. mori was the commonly used SpCas9, which only recognize target sites containing NGG PAM sequence. In the present study, we first improve the efficiency of our previous established SpCas9 system by 3.5 folds. The improved high efficiency was also observed at several loci in both BmNs cells and B. mori embryos. Then to expand the target sites, we showed that two newly discovered CRISPR system, SaCas9 and AsCpf1, could also induce highly efficient site-specific genome editing in BmNs cells, and constructed an integrated CRISPR system. Genome-wide analysis of targetable sites was further conducted and showed that the integrated system cover 69,144,399 sites in B. mori genome, and one site could be found in every 6.5 bp. The efficiency and resolution of this CRISPR platform will probably accelerate both fundamental researches and applicable studies in B. mori, and perhaps other insects.

  7. Expression of RYamide in the nervous and endocrine system of Bombyx mori.

    PubMed

    Roller, Ladislav; Čižmár, Daniel; Bednár, Branislav; Žitňan, Dušan

    2016-06-01

    RYamides are neuropeptides encoded by a gene whose precise expression and function have not yet been determined. We identified the RYamide gene transcript (fmgV1g15f, SilkBase database) and predicted two candidates for G-protein coupled RYamide receptors (A19-BAG68418 and A22-BAG68421) in the silkworm Bombyx mori. We cloned the RYamide transcript and described its spatial expression using in situ hybridisation. In the larval central nervous system (CNS) expression of RYamide was restricted to 12-14 small neurons in the brain and two posterior neurons in the terminal abdominal ganglion. During metamorphosis their number decreased to eight protocerebral neurons in the adults. Multiple staining, using various insect neuropeptide antibodies, revealed that neurons expressing RYamide are different from other peptidergic cells in the CNS. We also found RYamide expression in the enteroendocrine cells (EC) of the anterior midgut of larvae, pupae and adults. Two minor subpopulations of these EC were also immunoreactive to antibodies against tachykinin and myosupressin. This expression pattern suggests RYamides may play a role in the regulation of feeding and digestion. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Oxidative stress and spermatogenesis suppression in the testis of cadmium-treated Bombyx mori larvae.

    PubMed

    Yuan, Hongxia; Qin, Fenjv; Guo, Weiqiang; Gu, Huajie; Shao, Aihua

    2016-03-01

    Bombyx mori L. (B. mori) were exposed to cadmium chloride (CdCl2) incorporated in an artificial diet (0, 6.25, 12.5, 25, and 50 mg kg(-1)) throughout the larval stage. Changes in malondialdehyde (MDA) and reduced glutathione (GSH) contents and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), as well as their corresponding messenger RNA (mRNA) levels in the testes of the fifth instar larvae were evaluated. Additionally, spermatozoon deformation in the testes was examined. Upon Cd treatment, the MDA content in the testes was significantly increased in a concentration-dependent manner. Cd-exposed larvae had increased levels of glutathione. Pearson's correlation analysis revealed that SOD and CAT activities were positively correlated (R (2) = 0.605, P = 0.017). The changing trends in the mRNA levels of these enzymes were not always consistent with those of enzymatic activities. Alterations in GSH-Px activities and mRNA levels were positively correlated (R (2) = 0.771, P < 0.01). Morphological analysis revealed that Cd deformed and affected the maturation of spermatozoa. Our results collectively support a relationship between Cd and alterations in the levels of antioxidant enzymes in B. mori testes.

  9. High-level expression of canine parvovirus VP2 using Bombyx mori nucleopolyhedrovirus vector.

    PubMed

    Choi, J Y; Woo, S D; Lee, H K; Hong, H K; Je, Y H; Park, J H; Song, J Y; An, S H; Kang, S K

    2000-01-01

    For the potential use as recombinant vaccine, canine parvovirus (CPV) major capsid protein VP2 was expressed using Bombyx mori nucleopolyhedrovirus (BmNPV) vector. CPV VP2 gene was introduced into polyhedrin-based BmNPV transfer vector pBmKSK3, and recombinant virus BmK1-Parvo was prepared. When anti-CPV.VP2 monoclonal antibody was employed in immunofluorescence staining, an intense signal was observed within BmK1-Parvo-infected Bm5 cells but not within uninfected cells or cells infected with a wild-type BmNPV-K1. In hemagglutination assay, the expression level of VP2 were 3.2 x 10(3) HA units/ml from infected Bm5 cells, 2.1x 10(5) HA units/larvae from infected larval fat body, and 1.6x 10(6) HA units/ml from infected larval hemolymph. These results suggested that BmNPV vector system using B. mori larva as host could be applied to efficient mass-production of recombinant vaccines.

  10. Secretory mechanism of fibroin, a silk protein, in the posterior silk gland cells of Bombyx mori.

    PubMed

    Sasaki, S; Nakagaki, I

    1980-01-01

    There are two microtubule-microfilament systems in the posterior silk gland cells of Bombyx mori. One is a radial microtubule system; the other is a circular microtubule-microfilament system. These two systems are presumably concerned with the intracellular transport of secretory granules of fibroin and the secretion of fibroin into the lumen, respectively. Conventional and scanning electron microscopic observations of the two microtubule-microfilament systems in the posterior silk gland cells are reported. Scanning electron micrographs showed that a number of parallel linear cytoplasmic processes ran circularly on the luminal surface of the posterior silk gland cells. These processes were assumed to correspond to the circular microtubule-microfilament systems. The effects of cytochalasin (B or D), a secretion stimulating agent of fibroin, on the intracellular recording of membrane potential from the posterior silk gland cells are also reported. Exposure to cytochalasin resulted in depolarization of the membrane potential of the gland cells. Possible functional roles of the two microtubule-microfilament systems in the secretory mechanism of fibroin are discussed with reference to the effects of antimitotic reagents and cytochalasin on these two systems.

  11. Artificial parthenogenesis and control of voltinism to manage transgenic populations in Bombyx mori.

    PubMed

    Grenier, Anne-Marie; Da Rocha, Martine; Jalabert, Audrey; Royer, Corinne; Mauchamp, Bernard; Chavancy, Gérard

    2004-08-01

    In order to improve the management of transformed populations in a routine application of transgenesis technology in Bombyx mori, we modified its mode of reproduction and its voltinism. On one hand, after a stable integration of the gene of interest by transgenesis, it is preferable to maintain this gene in an identical genomic context through successive generations. This can be obtained by artificial parthenogenetic reproduction (ameiotic parthenogenesis) giving isogenic females identical to their transformed mother. On the other hand, it is essential to obtain continuous generations (polyvoltinism) after microinjection, in order to screen positive transgenic insects and study genetics and insertion of the transgene. Thereafter, it is more convenient to store these populations, as diapause eggs before their use in biotechnology application. We obtained such polyvoltine parthenoclones, first by selection for a parthenogenetic character in polyvoltine races, and second, by selection for a polyvoltine character in a parthenogenetic, but diapausing clone of B. mori. As diapause was directly under the control of diapause hormone (DH), we also tested direct injection of DH in female pupae of polyvoltine strains, as well as anti-DH antibody treatment to eliminate diapause in univoltine strains. We discussed the advantages and limitations of these methods and proved the feasibility in obtaining polyvoltine parthenoclones and determining the voltinism in B. mori. These methods would permit us to improve the management of populations used in transgenesis technology.

  12. Centuries of domestication has not impaired oviposition site-selection function in the silkmoth, Bombyx mori

    PubMed Central

    Damodaram, Kamala Jayanthi Pagadala; Kempraj, Vivek; Aurade, Ravindra Mahadappa; Rajasekhar, Sowmya Bandhisara; Venkataramanappa, Ravindra Kothapalli; Nandagopal, Bakthavatsalam; Verghese, Abraham

    2014-01-01

    Oviposition site-selection in insects is mediated through innate recognition templates (IRTs) tuned to specific chemical cues. These cues aid gravid insects in choosing suitable oviposition sites and may even enhance the fitness of their offspring by warding off predators and parasitoids. However, studies on the evolution of oviposition site-selection and cues instigating oviposition in domesticated insects remain elusive. Using the interaction between the silkmoth, Bombyx mori, and its host plant mulberry, Morus alba, as a model system, we demonstrate that centuries of domestication of silkmoth has not impaired its oviposition site-selection function. Silkmoths significantly preferred mulberry leaves to filter paper as oviposition sites. Oviposition assays with filter paper, filter paper treated with leaf volatiles and leaf alone proved that surface texture was not a significant criterion for oviposition site-selection, but volatile cues were. Oviposition assays with electrophysiologically active compounds from mulberry revealed that two of the volatiles, valencene and α-humulene, aided moths in choosing suitable oviposition sites and enhanced egg-laying significantly. Moreover, we show that generalist egg-parasitoids are strongly repelled by valencene and α-humulene. Our results demonstrate that IRTs tuned to cues that aid crucial functions like oviposition site-selection are less likely to be impaired even after centuries of domestication. PMID:25503440

  13. Genome-Wide Identification and Characterization of Carboxypeptidase Genes in Silkworm (Bombyx mori)

    PubMed Central

    Ye, Junhong; Li, Yi; Liu, Hua-Wei; Li, Jifu; Dong, Zhaoming; Xia, Qingyou; Zhao, Ping

    2016-01-01

    The silkworm (Bombyx mori) is an economically-important insect that can secrete silk. Carboxypeptidases have been found in various metazoan species and play important roles in physiological and biochemical reactions. Here, we analyzed the silkworm genome database and characterized 48 carboxypeptidases, including 34 metal carboxypeptidases (BmMCP1–BmMCP34) and 14 serine carboxypeptidases (BmSCP1–BmSCP14), to better understand their diverse functions. Compared to other insects, our results indicated that carboxypeptidases from silkworm have more family members. These silkworm carboxypeptidases could be divided into four families: Peptidase_M2 carboxypeptidases, Peptidase_M14 carboxypeptidases, Peptidase_S10 carboxypeptidases and Peptidase_S28 carboxypeptidases. Microarray analysis showed that the carboxypeptidases had distinct expression patterns, whereas quantitative real-time PCR demonstrated that the expression level of 13 carboxypeptidases significantly decreased after starvation and restored after re-feeding. Overall, our study provides new insights into the functional and evolutionary features of silkworm carboxypeptidases. PMID:27483237

  14. Seasonal variation in food consumption, assimilation, and conversion efficiency of Indian bivoltine hybrid silkworm, Bombyx mori.

    PubMed

    Rahmathulla, V K; Suresh, H M

    2012-01-01

    Food consumption and utilization is influenced by various biotic and abiotic factors. Under different environmental, feeding, and nutritional conditions, and with ingestion of the same amount of mulberry leaves, the silkworm shows significant difference in its ability to digest, absorb, and convert food to body matter. Here, influences of season, temperature, and humidity on food intake, assimilation, and conversion efficiency of the Indian bivoltine hybrid (CSR2 × CSR4) Bombyx mori L. (Lepidoptera: Bombycidae) were studied. The results indicated that food ingestion and assimilation were significantly higher among silkworm batches where optimum temperature and humidity were maintained compared with silkworm batches exposed to natural climatic conditions of the respective season. However, during summer the nutritional efficiency parameters were significantly higher among silkworms reared under natural temperature and humidity conditions when compared with the control. During the winter and rainy season, the nutritional efficiency parameters were significantly higher in control batches, where optimum temperature and humidity were maintained. Ingesta and digesta required to produce one gram of cocoon/shell were also lower in control batches for all seasons except summer. This may be due to the physiological adaptation of silkworms to overcome stress during the summer season.

  15. New insight into the mechanism underlying fibroin secretion in silkworm, Bombyx mori.

    PubMed

    Long, Dingpei; Lu, Weijian; Zhang, Yang; Guo, Qing; Xiang, Zhonghuai; Zhao, Aichun

    2015-01-01

    In order to investigate the role of different parts of the fibroin heavy chain (H-chain) in the secretion of fibroin in the silk gland of the silkworm (Bombyx mori) in vivo, two enhanced green fluorescent protein (EGFP)/H-chain fusion genes with deduced protein sequences containing an identical N-terminal region and different C-terminal regions of the H-chain were introduced into the B. mori genome using a piggyBac-mediated germline transformation. EGFP fluorescence and molecular analysis showed the products of two different EGFP/H-chain fusion proteins were secreted into the posterior silk gland lumen and aggregated in the middle silk gland and spun into cocoons. The results revealed that only the non-repetitive N terminus of the H-chain is essential for secretion of the H-chain into the posterior silk gland lumen. In addition, our results also indicated that the most likely post-translational modification of the H-chain is at the C-terminal domain. Here, our results not only provide a theoretical basis for the genetic modification of silk fiber as a functional biomaterial but also are of great significance to establishing a new silk gland bioreactor to mass-produce exogenous proteins in an active form. © 2014 FEBS.

  16. Genome-Wide Identification and Characterization of Carboxypeptidase Genes in Silkworm (Bombyx mori).

    PubMed

    Ye, Junhong; Li, Yi; Liu, Hua-Wei; Li, Jifu; Dong, Zhaoming; Xia, Qingyou; Zhao, Ping

    2016-07-28

    The silkworm (Bombyx mori) is an economically-important insect that can secrete silk. Carboxypeptidases have been found in various metazoan species and play important roles in physiological and biochemical reactions. Here, we analyzed the silkworm genome database and characterized 48 carboxypeptidases, including 34 metal carboxypeptidases (BmMCP1-BmMCP34) and 14 serine carboxypeptidases (BmSCP1-BmSCP14), to better understand their diverse functions. Compared to other insects, our results indicated that carboxypeptidases from silkworm have more family members. These silkworm carboxypeptidases could be divided into four families: Peptidase_M2 carboxypeptidases, Peptidase_M14 carboxypeptidases, Peptidase_S10 carboxypeptidases and Peptidase_S28 carboxypeptidases. Microarray analysis showed that the carboxypeptidases had distinct expression patterns, whereas quantitative real-time PCR demonstrated that the expression level of 13 carboxypeptidases significantly decreased after starvation and restored after re-feeding. Overall, our study provides new insights into the functional and evolutionary features of silkworm carboxypeptidases.

  17. Transcriptional Response of Silkworm (Bombyx mori) Eggs to O₂ or HCl Treatment.

    PubMed

    Gong, Jing; Tian, Sha; Zhou, Xia; Yang, Huan; Zhu, Yong; Hou, Yong

    2016-12-07

    Diapause is a common biological phenomenon that occurs in many organisms, including fish, insects, and nematodes. In the silkworm (Bombyx mori), diapause generally occurs in the egg stage. Treatment with O₂, HCl, or other compounds can prevent egg diapause. Here, we characterized the transcriptomic responses of newly laid eggs treated with O₂ or HCl. Digital gene expression analysis showed that 610 genes in O₂-treated eggs and 656 in HCl-treated eggs were differentially expressed. Of these, 343 genes were differentially expressed in both treatments. In addition to trehalases, sorbic acid dehydrogenases, and some enzymes involved in the carbohydrate metabolism, we also identified heat shock proteins, cytochrome P450, and GADD45, which are related to stress tolerance. Gene ontology enrichment analysis showed differentially expressed genes in O₂-treated eggs were involved in oxidoreductase activity as well as in binding, catalytic, and metabolic processes. The Kyoto Encyclopedia of Genes and Genomes analysis showed that the pathways for ribosome biogenesis, spliceosome, and circadian rhythm were significantly enriched in HCl-treated eggs. The reliability of the data was confirmed by qRT-PCR analysis. Our results improved the understanding of the mechanism of diapause blocking in silkworm eggs treated with O₂ or HCl and identified novel molecular targets for future studies.

  18. Can the silkworm (Bombyx mori) be used as a human disease model?

    PubMed

    Tabunoki, Hiroko; Bono, Hidemasa; Ito, Katsuhiko; Yokoyama, Takeshi

    2016-02-01

    Bombyx mori (silkworm) is the most famous lepidopteran in Japan. B. mori has long been used in the silk industry and also as a model insect for agricultural research. In recent years, B. mori has attracted interest in its potential for use in pathological analysis of model animals. For example, the human macular carotenoid transporter was discovered using information of B. mori carotenoid transporter derived from yellow-cocoon strain. The B. mori carotenoid transport system is useful in human studies. To develop a human disease model, we characterized the human homologs of B. mori, and by constructing KAIKO functional annotation pipeline, and to analyze gene expression profile of a unique B. mori mutant strain using microarray analysis. As a result, we identified a novel molecular network involved in Parkinson's disease. Here we describe the potential use of a spontaneous mutant silkworm strain as a human disease model. We also summarize recent progress in the application of genomic information for annotation of human homologs in B. mori. The B. mori mutant will provide a clue to pathological mechanisms, and the findings will be helpful for the development of therapies and for medical drug discovery.

  19. Molecular cloning, expression and characterization of acylpeptide hydrolase in the silkworm, Bombyx mori.

    PubMed

    Fu, Ping; Sun, Wei; Zhang, Ze

    2016-04-10

    Acylpeptide hydrolase (APH) can catalyze the release of the N-terminal amino acid from acetylated peptides. There were many documented examples of this enzyme in various prokaryotic and eukaryotic organisms. However, knowledge about APH in insects still remains unknown. In this study, we cloned and sequenced a putative silkworm Bombyx mori APH (BmAPH) gene. The BmAPH gene encodes a protein of 710 amino acids with a predicted molecular mass of 78.5kDa. The putative BmAPH and mammal APHs share about 36% amino acid sequence identity, yet key catalytic residues are conserved (Ser566, Asp654, and His686). Expression and purification of the recombinant BmAPH in Escherichia coli showed that it has acylpeptide hydrolase activity toward the traditional substrate, Ac-Ala-pNA. Furthermore, organophosphorus (OP) insecticides, chlorpyrifos, phoxim, and malathion, significantly inhibited the activity of the APH both in vitro and in vivo. In addition, BmAPH was expressed in all tested tissues and developmental stages of the silkworm. Finally, immunohistochemistry analysis showed that BmAPH protein was localized in the basement membranes. These results suggested that BmAPH may be involved in enhancing silkworm tolerance to the OP insecticides. In a word, our results provide evidence for understanding of the biological function of APH in insects. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Sexually dimorphic traits in the silkworm, Bombyx mori, are regulated by doublesex.

    PubMed

    Xu, Jun; Zhan, Shuai; Chen, Shuqing; Zeng, Baosheng; Li, Zhiqian; James, Anthony A; Tan, Anjiang; Huang, Yongping

    2017-01-01

    The DM domain genes, doublesex (dsx) in insects, or their structural homologs, male abnormal 3 (mab-3) in nematodes and Dmrt1 (doublesex and mab-3-related transcription factor 1) in mammals, are downstream regulators of the sex determination pathway that control sexually dimorphic development. Despite the functional importance of dsx and its potential applications in sterile insect technologies (SITs), the mechanisms by which it controls sexually dimorphic traits and the subsequent developmental gene networks in insects are poorly understood. Phylogenetic analyses indicate that insect dsx genes have sex-specific alternative splicing isoforms, whereas other taxa do not. We exploited genome editing and transgenesis technologies to induce mutations in either the male-specific isoform (dsx(M)) or common region (dsx(C)) of dsx in the somatic tissues of the lepidopteran model insect Bombyx mori. Disruptions of gene function produced either male-specific sexually-dimorphic defects or intersexual phenotypes; these results differ from those observed in other insects, including Drosophila melanogaster. Our data provide insights into the divergence of the insect sex determination pathways related to the most conserved downstream component dsx. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. BmncRNAdb: a comprehensive database of non-coding RNAs in the silkworm, Bombyx mori.

    PubMed

    Zhou, Qiu-Zhong; Zhang, Bindan; Yu, Quan-You; Zhang, Ze

    2016-09-13

    Long non-coding RNAs (lncRNAs) may play critical roles in a wide range of developmental processes of higher organisms. Recently, lncRNAs have been widely identified across eukaryotes and many databases of lncRNAs have been developed for human, mouse, fruit fly, etc. However, there is rare information about them in the only completely domesticated insect, silkworm (Bombyx mori). In this study, we systematically scanned lncRNAs using the available silkworm RNA-seq data and public unigenes. Finally, we identified and collected 6281 lncRNAs in the silkworm. Besides, we also collected 1986 microRNAs (miRNAs) from previous studies. Then, we organized them into a comprehensive and web-based database, BmncRNAdb. This database offers a user-friendly interface for data browse and online analysis as well as the three online tools for users to predict the target genes of lncRNA or miRNA. We have systematically identified and collected the silkworm lncRNAs and constructed a comprehensive database of the silkworm lncRNAs and miRNAs. This work gives a glimpse into lncRNAs of the silkworm and lays foundations for the ncRNAs study of the silkworm and other insects in the future. The BmncRNAdb is freely available at http://gene.cqu.edu.cn/BmncRNAdb/index.php .

  2. A comparison of virus genome sequences with their host silkworm, Bombyx mori.

    PubMed

    Tang, Xu-Dong; Yue, Ya-Jie; Wang, Wei; Li, Nan; Shen, Zhong-Yuan

    2016-01-15

    With the recent availability of the genomes of many viruses and the silkworm, Bombyx mori, as well as a variety of Basic Local Alignment Search Tool (BLAST) programs, a new opportunity to gain insight into the interaction of viruses with the silkworm is possible. This study aims to determine the possible existence of sequence identities between the genomes of viruses and the silkworm and attempts to explain this phenomenon. BLAST searches of the genomes of viruses against the silkworm genome were performed using the resources of the National Center for Biotechnology Information. All studied viruses contained variable numbers of short regions with sequence identity to the genome of the silkworm. The short regions of sequence identity in the genome of the silkworm may be derived from the genomes of viruses in the long history of silkworm-virus interaction. This study is the first to compare these genomes, and may contribute to research on the interaction between viruses and the silkworm. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Effects of different Bombyx mori silkworm varieties on the structural characteristics and properties of silk.

    PubMed

    Chung, Da Eun; Kim, Hyung Hwan; Kim, Moo Kon; Lee, Ki Hoon; Park, Young Hwan; Um, In Chul

    2015-08-01

    Silk has attracted the attention of biomedical researchers because of its good biocompatibility. Although various characteristics of silk are needed for its successful application in biomedical fields, the performance of silk material is limited. Although there are many varieties of Bombyx mori silkworm, the effect of different silkworm varieties on regenerated silk has not been considered in detail. That is, the use of a diverse variety of silkworms has not been considered in non-textile applications resulting in limited performance of silk materials. In this study, the effects of different silkworm varieties on the structural characteristics and properties of silk cocoon and regenerated silk fibroin (SF) were examined. Structural characteristics of silk cocoon including color, fiber diameter, and porosity, differed depending on the silkworm variety. Furthermore, molecular weight, solution viscosity, and mechanical properties of regenerated SF were influenced by the variety of silkworm, while the amino acid composition, β-sheet crystallization by formic acid, and cyto-compatibility of regenerated SF did not differ between the samples from different varieties of silkworm. These results imply that diverse performance of silk can be obtained by controlling the silkworm variety, and that the use of different varieties of silkworm might be a good way to strengthen the performance of silk in biomedical fields. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Analysis of the activity of virus internal ribosome entry site in silkworm Bombyx mori.

    PubMed

    Ye, Lupeng; Zhuang, Lanfang; Li, Jisheng; You, Zhengying; Liang, Jianshe; Wei, Hao; Lin, Jianrong; Zhong, Boxiong

    2013-07-01

    Internal ribosome entry site (IRES) has been widely used in genetic engineering; however, the application in silkworm (Bombyx mori) has hardly been reported. In this study, the biological activity of partial sequence of Encephalomyocarditis virus (EMCV) IRES, Rhopalosiphum padi virus (RhPV) IRES, and the hybrid of IRES of EMCV and RhPV were investigated in Spodoptera frugiperda (Sf9) cell line and silkworm tissues. The hybrid IRES of EMCV and RhPV showed more effective than EMCV IRES or RhPV IRES in promoting downstream gene expression in insect and silkworm. The activities of all IRESs in middle silk gland of silkworm were higher than those in the fat body and posterior silk gland. The hybrid IRES of EMCV and RhPV was integrated into silkworm genome by transgenic technology to test biological activity of IRES. Each of the positive transgenic individuals had significant expression of report gene EGFP. These results suggested that IRES has a potential to be used in the genetic engineering research of silkworm.

  5. Transcriptome analysis of silkworm, Bombyx mori, during early response to Beauveria bassiana challenges.

    PubMed

    Hou, Chengxiang; Qin, Guangxing; Liu, Ting; Geng, Tao; Gao, Kun; Pan, Zhonghua; Qian, Heying; Guo, Xijie

    2014-01-01

    Host-pathogen interactions are complex processes and it is a central challenge to reveal these interactions. Fungal infection of silkworm, Bombyx mori, may induce a variety of responsive reaction. However, little is known about the molecular mechanism of silkworm immune response against the fungal infection. To obtain an overview of the interaction between silkworm and an entomopathogenic fungus Beauveria bassiana, Digital Gene Expression profiling, a tag based high-throughput transcriptome sequencing method, was employed to screen and identify differentially expressed genes (DEGs, FDR ≤ 0.001, ∣log2ratio∣ ≥ 1) of silkworm larvae during early response against B. bassiana infection. Total 1430 DEGs including 960 up-regulated and 470 down-regulated ones were identified, of which 627 DEGs can be classified into GO categories by Gene Ontology (GO) analysis. KEGG pathways analysis of these DEGs suggested that many biological processes, such as defense and response, signal transduction, phagocytosis, regulation of gene expression, RNA splicing, biosynthesis and metabolism, protein transport etc. were involved in the interaction between the silkworm and B. bassiana. A number of differentially expressed fungal genes were also identified by mapping the sequencing tags to B. bassiana genome. These results provided new insights to the molecular mechanism of silkworm immune response to B. bassiana infection.

  6. Molecular cloning and analysis of a C-type lectin from silkworm Bombyx mori.

    PubMed

    Shahzad, Toufeeq; Zhan, Ming-Yue; Yang, Pei-Jin; Yu, Xiao-Qiang; Rao, Xiang-Jun

    2017-07-01

    C-type lectins (CTLs) play a variety of roles in plants and animals. They are involved in animal development, pathogen recognition, and the activation of immune responses. CTLs carry one or more non-catalytic carbohydrate-recognition domains (CRDs) to bind specific carbohydrates reversibly. Here, we report the molecular cloning and functional analysis of a single-CRD CTL, named C-type lectin-S2 (BmCTL-S2) from the domesticated silkmoth Bombyx mori (Lepidoptera: Bombycidae). The ORF of CTL-S2 is 666 bp, which encodes a putative protein of 221 amino acids. BmCTL-S2 is expressed in a variety of immune-related tissues, including hemocytes and fat body among others. BmCTL-S2 mRNA level in the midgut and the fat body was significantly increased by bacterial challenges. The recombinant protein (rBmCTL-S2) bound different bacterial cell wall components and bacterial cells. rBmCTL-S2 also inhibited the growth of Bacillus subtilis and Staphylococcus aureus. Taken together, we infer that BmCTL-S2 is a pattern-recognition receptor with antibacterial activities. © 2017 Wiley Periodicals, Inc.

  7. Developmental Changes for the Hemolymph Metabolome of Silkworm (Bombyx mori L.).

    PubMed

    Zhou, Lihong; Li, Huihui; Hao, Fuhua; Li, Ning; Liu, Xin; Wang, Guoliang; Wang, Yulan; Tang, Huiru

    2015-05-01

    Silkworm (Bombyx mori) is a lepidopteran-holometabolic model organism. To understand its developmental biochemistry, we characterized the larval hemolymph metabonome from the third instar to prepupa stage using (1)H NMR spectroscopy whilst hemolymph fatty acid composition using GC-FID/MS. We unambiguously assigned more than 60 metabolites, among which tyrosine-o-β-glucuronide, mesaconate, homocarnosine, and picolinate were reported for the first time from the silkworm hemolymph. Phosphorylcholine was the most abundant metabolite in all developmental stages with exception for the periods before the third and fourth molting. We also found obvious developmental dependence for the hemolymph metabonome involving multiple pathways including protein biosyntheses, glycolysis, TCA cycle, the metabolisms of choline amino acids, fatty acids, purines, and pyrimidines. Most hemolymph amino acids had two elevations during the feeding period of the fourth instar and prepupa stage. Trehalose was the major blood sugar before day 8 of the fifth instar, whereas glucose became the major blood sugar after spinning. C16:0, C18:0 and its unsaturated forms were dominant fatty acids in hemolymph. The developmental changes of hemolymph metabonome were associated with dietary nutrient intakes, biosyntheses of cell membrane, pigments, proteins, and energy metabolism. These findings offered essential biochemistry information in terms of the dynamic metabolic changes during silkworm development.

  8. The progress and future of enhancing antiviral capacity by transgenic technology in the silkworm Bombyx mori.

    PubMed

    Jiang, Liang; Xia, Qingyou

    2014-05-01

    Bombyx mori is a common lepidopteran model and an important economic insect for silk production. B. mori nucleopolyhedrovirus (BmNPV) is a typical pathogenic baculovirus that causes serious economic losses in sericulture. B. mori and BmNPV are a model of insect host and pathogen interaction including invasion of the host by the pathogen, host response, and enhancement of host resistance. The antiviral capacity of silkworms can be improved by transgenic technology such as overexpression of an endogenous or exogenous antiviral gene, RNA interference of the BmNPV gene, or regulation of the immune pathway to inhibit BmNPV at different stages of infection. Antiviral capacity could be further increased by combining different methods. We discuss the future of an antiviral strategy in silkworm, including possible improvement of anti-BmNPV, the feasibility of constructing transgenic silkworms with resistance to multiple viruses, and the safety of transgenic silkworms. The silkworm model could provide a reference for disease control in other organisms. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. Vertebrate estrogen regulates the development of female characteristics in silkworm, Bombyx mori.

    PubMed

    Shen, Guanwang; Lin, Ying; Yang, Congwen; Xing, Runmiao; Zhang, Haiyan; Chen, Enxiang; Han, Chaoshan; Liu, Hongling; Zhang, Weiwei; Xia, Qingyou

    2015-01-01

    The vertebrate estrogens include 17-β-estradiol (E2), which has an analog in silkworm ovaries. In this study, the Bombyx mori vitellogenin gene (BmVg) was used as a biomarker to analyze the function of the E2 in silkworm. In most oviparous animals, Vg has female-specific expression. However, BmVg expression was also detected in B. mori males. Stage specific fluctuation of BmVg expression was similar in males and females, but expression levels in males were lower than in females. E2 treatment by injection or feeding of male larvae in the final instar stage induced and stimulated male BmVg transcription and protein synthesis. When silkworm ovary primordia were transplanted into males, BmVg was induced in male fat bodies. Transplanted ovaries primordia were also able to develop into ovaries and produce mature eggs. When females were treated with E2 promoted BmVg/BmVn protein accumulation in hemolymph, ovaries and eggs. However, BmVg transcription was decreased in female fat bodies. An E2 analog was identified in the hemolymph of day 3 wandering silkworms using high-performance liquid chromatography. Estradiol titers from fifth late-instar larvae to pupal stage were determined by enzyme-linked immunosorbent assay. The results suggested that silkworms synthesized a vertebrate E2 analog. This study found that E2 promoted the synthesis of BmVg, a female typical protein in silkworms. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Melanin pigmentation gives rise to black spots on the wings of the silkworm Bombyx mori.

    PubMed

    Ito, Katsuhiko; Yoshikawa, Manabu; Fujii, Takeshi; Tabunoki, Hiroko; Yokoyama, Takeshi

    2016-01-01

    Several mutants of the silkworm Bombyx mori show body color variation at the larval and adult stages. The Wild wing spot (Ws) mutant exhibits a phenotype in which the moth has a spot on the apex of the forewing. In this study, we investigated this trait to elucidate the molecular mechanism underlying the color pattern. Microscopy of the black spot of Ws mutants showed that the pigment emerges in the scales of the wing, and accumulation of the pigment becomes strong just before eclosion. We next examined the relationship between the black spot of the Ws mutant and melanin. The spectrophotometry using alkaline extracts from the black spot in the wing showed the highest absorption intensity at 405nm, which is the absorbance wavelength of melanin. Moreover, inhibition assays for enzymes implicated in melanin synthesis using 3-iodo-l-tyrosine (a tyrosine hydroxylase inhibitor) and L-α-methyl-DOPA (a dopa decarboxylase inhibitor) revealed that treatment with each inhibitor disrupted the pigmentation of the wing of the Ws mutant. On the basis of these results, we analyzed the expression pattern of five genes involved in melanin formation, and found that the expression levels of yellow and laccase2 were increased just before pigmentation, whereas those of DDC, tan, and TH were increased when the apex of the wing turned black. These results showed that melanin pigmentation gives rise to the black spot on the wing. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Characterization and identification of the integrin family in silkworm, Bombyx mori.

    PubMed

    Zhang, Kui; Xu, Man; Su, Jingjing; Yu, Shuang; Sun, Zhongfeng; Li, Yutian; Zhang, Weibo; Hou, Jianbing; Shang, Lijun; Cui, Hongjuan

    2014-10-01

    As an important economic insect, Bombyx mori is also a useful model organism for lepidopteran insect. Integrins are evolutionarily conserved from sponges to humans, and play vital roles in many physiological and pathological processes. To explore their diverse functions of integrins in insect, eleven integrins including six α and five β subunits were cloned and characterized from silkworm. Our results showed that integrins from silkworm own more family members compared to other invertebrates. Among those α subunits, integrins α1, α2, and the other four subunits belong to PS1, PS2, and PS3 groups, respectively. The β subunits mainly gather in the insect βν group except the β1 subunit which belongs to the insect β group. Expression profiles demonstrated that the integrins exhibited distinct patterns, but were mainly expressed in hemocytes. α1 and β2 subunits are the predominant ones either in the embryogenesis or larva stages. Interestingly, integrins were significantly up-regulated after stimulated by 20-hydroxyecdysone (20-E) in vivo. These results indicate that integrins perform diverse functions in hemocytes of silkworm. Overall, our results provide a new insight into the functional and evolutionary features of integrins. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Functional Loss of Bmsei Causes Thermosensitive Epilepsy in Contractile Mutant Silkworm, Bombyx mori

    PubMed Central

    Nie, Hongyi; Cheng, Tingcai; Huang, Xiaofeng; Zhou, Mengting; Zhang, Yinxia; Dai, Fangyin; Mita, Kazuei; Xia, Qingyou; Liu, Chun

    2015-01-01

    The thermoprotective mechanisms of insects remain largely unknown. We reported the Bombyx mori contractile (cot) behavioral mutant with thermo-sensitive seizures phenotype. At elevated temperatures, the cot mutant exhibit seizures associated with strong contractions, rolling, vomiting, and a temporary lack of movement. We narrowed a region containing cot to ~268 kb by positional cloning and identified the mutant gene as Bmsei which encoded a potassium channel protein. Bmsei was present in both the cell membrane and cytoplasm in wild-type ganglia but faint in cot. Furthermore, Bmsei was markedly decreased upon high temperature treatment in cot mutant. With the RNAi method and injecting potassium channel blockers, the wild type silkworm was induced the cot phenotype. These results demonstrated that Bmsei was responsible for the cot mutant phenotype and played an important role in thermoprotection in silkworm. Meanwhile, comparative proteomic approach was used to investigate the proteomic differences. The results showed that the protein of Hsp-1 and Tn1 were significantly decreased and increased on protein level in cot mutant after thermo-stimulus, respectively. Our data provide insights into the mechanism of thermoprotection in insect. As cot phenotype closely resembles human epilepsy, cot might be a potential model for the mechanism of epilepsy in future. PMID:26198671

  13. Seasonal Variation in Food Consumption, Assimilation, and Conversion Efficiency of Indian Bivoltine Hybrid Silkworm, Bombyx mori

    PubMed Central

    Rahmathulla, V. K.; Suresh, H. M.

    2012-01-01

    Food consumption and utilization is influenced by various biotic and abiotic factors. Under different environmental, feeding, and nutritional conditions, and with ingestion of the same amount of mulberry leaves, the silkworm shows significant difference in its ability to digest, absorb, and convert food to body matter. Here, influences of season, temperature, and humidity on food intake, assimilation, and conversion efficiency of the Indian bivoltine hybrid (CSR2 × CSR4) Bombyx mori L. (Lepidoptera: Bombycidae) were studied. The results indicated that food ingestion and assimilation were significantly higher among silkworm batches where optimum temperature and humidity were maintained compared with silkworm batches exposed to natural climatic conditions of the respective season. However, during summer the nutritional efficiency parameters were significantly higher among silkworms reared under natural temperature and humidity conditions when compared with the control. During the winter and rainy season, the nutritional efficiency parameters were significantly higher in control batches, where optimum temperature and humidity were maintained. Ingesta and digesta required to produce one gram of cocoon/shell were also lower in control batches for all seasons except summer. This may be due to the physiological adaptation of silkworms to overcome stress during the summer season. PMID:23414194

  14. Expression, purification and characterization of an atypical 2-Cys peroxiredoxin from the silkworm, Bombyx mori.

    PubMed

    Zhang, L; Lu, Z

    2015-04-01

    Peroxiredoxins (Prxs) play important roles in protecting organisms against damage caused by reactive oxygen species (ROS). In this study, we cloned a cDNA of Bombyx mori peroxiredoxin 5 (BmPrx5), which contained a 565-bp open reading frame for a 188-residue protein. Sequence analysis indicated that BmPrx5 belongs to the atypical 2-Cys peroxiredoxin family. Recombinant BmPrx5 purified from Escherichia coli showed antioxidant activity that removes H2 O2 and protects DNA from oxidative damage. Quantitative real-time PCR showed that the level of BmPrx5 mRNA in haemocytes increased early and decreased by 24 h after injection of H2 O2 whereas, in the fat body, the transcript level decreased at 6 h and increased at 12 h. Pseudomonas aeruginosa and Staphylococcus aureus infection resulted in higher levels of H2 O2 in the haemolymph and of BmPrx5 mRNA in haemocytes at 8 h postinfection. These data suggest that BmPrx5 acts as an antioxidant enzyme to protect the silkworm from oxidative damage induced by bacterial infection. Further study is needed to elucidate the exact role of BmPrx5 in the silkworm immune system. © 2014 The Royal Entomological Society.

  15. Identification and characterization of an atypical 2-cys peroxiredoxin from the silkworm, Bombyx mori.

    PubMed

    Wang, Q; Zhou, Y; Chen, K; Ju, X

    2016-08-01

    Peroxiredoxins (Prxs) play an important role in the protection of insects against the toxicity of reactive oxygen species. Here, we identified and characterized a novel, atypical 2-cysteine (Cys) peroxiredoxin (BmPrx3) from an expressed sequence tag database in a lepidopteran insect, Bombyx mori. The BmPrx3 cDNA contained an open reading frame of 684 bp that encodes a 228-amino-acid protein with a calculated molecular mass of 25 kDa. Sequence comparison revealed that BmPrx3 belongs to the atypical 2-Cys Prxs. Quantitative real-time PCR revealed that BmPrx3 can be detected in all tissues and developmental stages. Recombinant BmPrx3 purified from Escherichia coli exhibited antioxidant activity that removed hydrogen peroxide and protected DNA from oxidative damage. Disc diffusion and viability assays revealed that recombinant BmPrx3 increased bacterial survival under H2 O2 -mediated oxidative stress. In addition, quantitative real-time PCR analysis indicated that BmPrx3 transcription levels were significantly increased in response to various oxidative stresses. Furthermore, BmPrx3 transcription levels in the midgut were regulated by bacterial infection. Taken together, these results suggest that BmPrx3 acts as an antioxidant enzyme to protect the silkworm from various oxidative stresses. © 2016 The Royal Entomological Society.

  16. Genome-wide identification and characterization of Fox genes in the silkworm, Bombyx mori.

    PubMed

    Song, JiangBo; Li, ZhiQuan; Tong, XiaoLing; Chen, Cong; Chen, Min; Meng, Gang; Chen, Peng; Li, ChunLin; Xin, YaQun; Gai, TingTing; Dai, FangYin; Lu, Cheng

    2015-09-01

    The forkhead box (Fox) transcription factor family has a characteristic of forkhead domain, a winged DNA-binding domain. The Fox genes have been classified into 23 subfamilies, designated FoxA to FoxS, of which the FoxR and FoxS subfamilies are specific to vertebrates. In this review, using whole-genome scanning, we identified 17 distinct Fox genes distributed on 13 chromosomes of the silkworm, Bombyx mori. A phylogenetic tree showed that the silkworm Fox genes could be classified into 13 subfamilies. The FoxK subfamily is specifically absent from the silkworm, although it is present in other lepidopteran insects, including Danaus plexippus and Heliconius melpomene. Microarray data revealed that the Fox genes have distinct expression patterns in the tissues on day 3 of the 5th instar larva. A Gene Ontology analysis suggested that the Fox genes have roles in cellular components, molecular functions, and biological processes, except in pore complex biogenesis. An analysis of the selective pressure on the proteins indicated that most of the amino acid sites in the Fox proteins are undergoing strong purifying selection. Here, we summarize the general characteristics of the Fox genes in the silkworm, which should support further functional studies of the silkworm Fox proteins.

  17. FLP Recombinase-Mediated Site-Specific Recombination in Silkworm, Bombyx mori

    PubMed Central

    Long, Ding-Pei; Zhao, Ai-Chun; Chen, Xue-Jiao; Zhang, Yang; Lu, Wei-Jian; Guo, Qing; Handler, Alfred M.; Xiang, Zhong-Huai

    2012-01-01

    A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species. PMID:22768245

  18. Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

    SciTech Connect

    Kajikawa, Mizuho; Sasaki, Kaori; Wakimoto, Yoshitaro; Toyooka, Masaru; Motohashi, Tomoko; Shimojima, Tsukasa; Takeda, Shigeki; Park, Enoch Y.; Maenaka, Katsumi

    2009-07-31

    Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G{sub i}{alpha}) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [{sup 35}S]GTP{gamma}S-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.

  19. Enhancing effect of glycerol on the tensile properties of Bombyx mori cocoon sericin films.

    PubMed

    Zhang, Haiping; Deng, Lianxia; Yang, Mingying; Min, Sijia; Yang, Lei; Zhu, Liangjun

    2011-01-01

    An environmental physical method described herein was developed to improve the tensile properties of Bombyx mori cocoon sericin films, by using the plasticizer of glycerol, which has a nontoxic effect compared with other chemical crosslinkers. The changes in the tensile characteristics and the structure of glycerolated (0-40 wt% of glycerol) sericin films were investigated. Sericin films, both in dry and wet states, showed enhanced tensile properties, which might be regulated by the addition of different concentrations of glycerol. The introduction of glycerol results in the higher amorphous structure in sericin films as evidenced by analysis of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra, thermogravimetry (TGA) and differential scanning calorimetry (DSC) curves. Scanning Electron Microscopy (SEM) observation revealed that glycerol was homogeneously blended with sericin molecules when its content was 10 wt%, while a small amount of redundant glycerol emerged on the surface of sericin films when its content was increased to 20 wt% or higher. Our results suggest that the introduction of glycerol is a novel nontoxic strategy which can improve the mechanical features of sericin-based materials and subsequently promote the feasibility of its application in tissue engineering.

  20. Identification of a new Bombyx mori nucleopolyhedrovirus and analysis of its bro gene family.

    PubMed

    Zhou, Jing-Bo; Li, Xing-Qi; De-Eknamkul, Wanchai; Suraporn, Siripuk; Xu, Jia-Ping

    2012-06-01

    The highly pathogenic Bombyx mori nucleopolyhedrovirus (BmNPV) has caused severe damages to sericulture in many countries, and the relationship between the pathogenicity of various BmNPV strains and their geographical evolution has been the topic of our interest. In this study, we isolated a new BmNPV strain from Thailand (BmNPV-Thai), based on the sequences of its conservative genes p10, p35, polh, egt and vp39. The BmNPV-Thai appears to have baculovirus repeated ORF (bro) genes different from four other well-known BmNPV strains of China (GD, CQ1), Japan (T3), and France (SC7); It only has bro-a, bro-c, and bro-d, but not bro-b and bro-e genes. These bro genes are localized only in the two subgroups highly homologous to their counterparts and their encoded BRO proteins differ mainly at their N-terminal amino acid residues. Phylogenetic analysis indicates that the evolution of the bro genes of the five BmNPV strains is not obviously associated with their geographic locations.

  1. Diversity, distribution, and mobility of bro gene sequences in Bombyx mori nucleopolyhedrovirus.

    PubMed

    López Ferber, M; Argaud, O; Croizier, L; Croizier, G

    2001-06-01

    Genetic differences between strains of a baculovirus are often limited to some restriction sites, short DNA deletions or absence of some nonessential genes. The recently coined bro gene family, represents a new major source of intraspecific variability. A comparison between two bro gene sets of Bombyx mori nucleopolyhedroviruses (NPV) shows that bro genes are distributed in three regions for the -T3 and -SC7 virus strains. In BmNPV T3, five bro genes are distributed in three genome locations, whereas the BmNPV SC7 strain possess a single bro copy in each region. In addition, each of the BmNPV SC7 bro genes belongs to one of the three subfamilies present in BmNPV T3. Analysis of bro copy sequences and of adjacent sequences suggests an active redistribution of sequences due to intraspecific recombination. The maintenance of one allele of each subfamily suggests that they play different roles in the viral cycle, and that they are essential.

  2. Changes in glutathione redox cycle during diapause determination and termination in the bivoltine silkworm, Bombyx mori.

    PubMed

    Zhao, Lin-Chuan; Hou, Yi-Sheng; Sima, Yang-Hu

    2014-02-01

    To explore whether glutathione regulates diapause determination and termination in the bivoltine silkworm Bombyx mori, we monitored the changes in glutathione redox cycle in the ovary of both diapause- and nondiapause-egg producers, as well as those in diapause eggs incubated at different temperatures. The activity of thioredoxin reductase (TrxR) was detected in ovaries but not in eggs, while neither ovaries nor eggs showed activity of glutathione peroxidase. A lower reduced glutathione/oxidized glutathione (GSH/GSSG) ratio was observed in the ovary of diapause-egg producers, due to weaker reduction of oxidized glutathione (GSSG) to the reduced glutathione (GSH) catalyzed by glutathione reductase (GR) and TrxR. This indicates an oxidative shift in the glutathione redox cycle during diapause determination. Compared with the 25°C-treated diapause eggs, the 5°C-treated diapause eggs showed lower GSH/GSSG ratio, a result of stronger oxidation of GSH catalyzed by thioredoxin peroxidase and weaker reduction of GSSG catalyzed by GR. Our study demonstrated the important regulatory role of glutathione in diapause determination and termination of the bivoltine silkworm.

  3. Aspartate Decarboxylase is Required for a Normal Pupa Pigmentation Pattern in the Silkworm, Bombyx mori

    PubMed Central

    Dai, Fangyin; Qiao, Liang; Cao, Cun; Liu, Xiaofan; Tong, Xiaoling; He, Songzhen; Hu, Hai; Zhang, Li; Wu, Songyuan; Tan, Duan; Xiang, Zhonghuai; Lu, Cheng

    2015-01-01

    The pigmentation pattern of Lepidoptera varies greatly in different development stages. To date, the effects of key genes in the melanin metabolism pathway on larval and adult body color are distinct, yet the effects on pupal pigmentation remains unclear. In the silkworm, Bombyx mori, the black pupa (bp) mutant is only specifically melanized at the pupal stage. Using positional cloning, we found that a mutation in the Aspartate decarboxylase gene (BmADC) is causative in the bp mutant. In the bp mutant, a SINE-like transposon with a length of 493 bp was detected ~2.2 kb upstream of the transcriptional start site of BmADC. This insertion causes a sharp reduction in BmADC transcript levels in bp mutants, leading to deficiency of β-alanine and N-β-alanyl dopamine (NBAD), but accumulation of dopamine. Following injection of β-alanine into bp mutants, the color pattern was reverted that of the wild-type silkworms. Additionally, melanic pupae resulting from knock-down of BmADC in the wild-type strain were obtained. These findings show that BmADC plays a crucial role in melanin metabolism and in the pigmentation pattern of the silkworm pupal stage. Finally, this study contributes to a better understanding of pupa pigmentation patterns in Lepidoptera. PMID:26077025

  4. Identification and Molecular Characterization of a Chitin Deacetylase from Bombyx mori Peritrophic Membrane

    PubMed Central

    Zhong, Xiao-Wu; Wang, Xiao-Huan; Tan, Xiang; Xia, Qing-You; Xiang, Zhong-Huai; Zhao, Ping

    2014-01-01

    The insect midgut epithelium is generally lined with a unique chitin and protein structure, the peritrophic membrane (PM), which facilitates food digestion and protects the gut epithelium. PM proteins are important determinants for PM structure and formation. In this study, the silkworm Bombyx mori midgut PM protein BmCDA7 was identified by proteomic tools. The full-length BmCDA7 cDNA is 1357 bp; the deduced protein is composed of 379 amino acid residues and includes a 16 amino acid residue signal peptide, a putative polysaccharide deacetylase-like domain and 15 cysteine residues present in three clusters. The heterologously expressed proteins of the BmCDA7 gene in yeast displayed chitin deacetylase activity. Expression of B. mori BmCDA7 was detected in the midgut at both the transcriptional and translational levels. The BmCDA7 gene was expressed by the newly hatched silkworm larvae until day seven of the fifth instar and was expressed at a high level in the newly exuviated larvae of different instars. The functions and regulatory mechanism of BmCDA7, however, need further investigation. PMID:24473143

  5. Participation of D-serine in the development and reproduction of the silkworm Bombyx mori.

    PubMed

    Tanigawa, Minoru; Suzuki, Chihiro; Niwano, Kimio; Kanekatsu, Rensuke; Tanaka, Hiroyuki; Horiike, Kihachiro; Hamase, Kenji; Nagata, Yoko

    2016-04-01

    The silkworm Bombyx mori contains high concentrations of free D-serine, an optical isomer of L-serine. To elucidate its function, we first investigated the localization of D-serine in various organs of silkworm larvae, pupae, and adult moths. Using immunohistochemical analysis with an anti-D-serine antibody, we found D-serine in the microvilli of midgut goblet and cylindrical cells and in peripheral matrix components of testicular and ovarian cells. By spectrophotometric analysis, D-serine was also found in the hemolymph and fat body. D-Alanine was not detected in the various organs by immunohistochemistry. Serine racemase, which catalyzes the inter-conversion of L- and D-serine, was found to co-localize with D-serine, and D-serine production from L-serine by intrinsic serine racemase was suggested. O-Phospho-L-serine is an inhibitor of serine racemase, and it was administered to the larvae to reduce the D-serine level. This reagent decreased the midgut caspase-3 level and caused a delay in spermatogenesis and oogenesis. The reagent also decreased mature sperm and egg numbers, suggesting D-serine participation in these processes. D-Serine administration induced an increase in pyruvate levels in testis, midgut, and fat body, indicating conversion of D-serine to pyruvate. On the basis of these results, together with our previous investigation of ATP biosynthesis in testis, we consider the possible involvement of D-serine in ATP synthesis for metamorphosis and reproduction.

  6. Proteomics analysis of digestive juice from silkworm during Bombyx mori nucleopolyhedrovirus infection.

    PubMed

    Hu, Xiaolong; Zhu, Min; Wang, Simei; Zhu, Liyuan; Xue, Renyu; Cao, Guangli; Gong, Chengliang

    2015-08-01

    Previous studies have analyzed the midgut transcriptome and proteome after challenge with Bombyx mori nucleopolyhedrovirus (BmNPV), however little information is available on the digestive juice proteome after BmNPV challenge. This study investigated BmNPV infection-induced protein changes in the digestive juice of silkworms using shotgun proteomics and MS sequencing. From the digestive juice of normal third-day, fifth-instar silkworm larvae, 75 proteins were identified, 44 of which were unknown; from larvae 6 h after inoculation with BmNPV, 106 proteins were identified, of which 39 were unknown. After BmNPV challenge, more secreted proteins appeared that had antiviral and digestive features. GO annotation analysis clustered most proteins in the lumen into catalytic, binding, and metabolic processes. Numerous proteins were reported to have BmNPV interactions. Hsp70 protein cognate, lipase-1, and chlorophyllide A-binding protein precursor were upregulated significantly after BmNPV challenge. Levels of trypsin-like serine protease, beta-1,3-glucanase, catalase, and serine protease transcripts decreased or were not significantly change after BmNPV challenge. Taken together, these findings provided insights into the interaction between host and BmNPV and revealed potential functions of digestive juice after per os BmNPV infection.

  7. Molecular and Physiological Characterization of Two Novel Multirepeat β-Thymosins from Silkworm, Bombyx mori.

    PubMed

    Ma, Shangshang; Kang, Zhiqiong; Lü, Peng; Yang, Yanhua; Yao, Qin; Xia, Hengchuan; Chen, Keping

    2015-01-01

    β-thymosin plays important roles in the development of the lymphatic system and the central nervous system in vertebrates. However, its role and function in invertebrates remain much less explored. Here, we firstly isolated a gene encoding β-thymosin in silkworm (Bombyx mori L.). Interestingly, this gene encodes two polypeptides, named as BmTHY1 and BmTHY2, via two different modes of RNA splicing. The recombinant proteins fused with an N-term GST tag were over-expressed in Escherichia coli (E. coli) and further purified to near homogenity to prepare mouse antibodies. The Western blot analysis showed that these proteins were expressed in various tissues and organs, as well as in different developmental stages. Amazingly, the expression of BmTHY2 was hugely increased during the pupae stage, indicating a specialized role in this period. The expression of these proteins was gradually decreased in BmN cells infected by BmNPV, suggesting they may play different roles in the virus infection. In addition, both BmTHY1 and BmTHY2 can interact with 14-3-3 of silkworm and Ubiquitin of BmNPV as shown by GST pull down and Co-IP assays, consistent with their roles in the regulation of the development of nervous system.

  8. Characterization of a dual-CRD galectin in the silkworm Bombyx mori.

    PubMed

    Rao, Xiang-Jun; Wu, Peng; Shahzad, Toufeeq; Liu, Su; Chen, Ling; Yang, Yun-Fan; Shi, Qiao; Yu, Xiao-Qiang

    2016-07-01

    Galectins (S-type lectins) are an ancient family of lectins with the β-galactoside binding activity. In mammals, galectins play essential roles in many biological processes, such as development, immune homeostasis and tumor progression. However, few studies have been devoted to their functions in insects. Here, we characterized the only dual-CRD galectin in the silkworm Bombyx mori (BmGalectin-4). BmGalectin-4 cDNA possesses an open reading frame of 1089 bp, which encodes a putative galectin of 363 amino acids containing tandem carbohydrate recognition domains (CRDs). BmGalectin-4 was expressed in various tissues but the protein was most abundant in fertilized eggs. Its transcript level in fertilized eggs was upregulated upon bacterial challenge. Recombinant BmGalectin-4 purified from Escherichia coli bound to bacterial cell wall components and bacterial cells. In addition, the recombinant protein induced bacterial agglutination, but did not have antibacterial activity against selected microorganisms. Taken together, our results suggest that BmGalectin-4 may function as a pattern recognition receptor primarily in silkworm fertilized eggs.

  9. Advanced technologies for genetically manipulating the silkworm Bombyx mori, a model Lepidopteran insect.

    PubMed

    Xu, Hanfu; O'Brochta, David A

    2015-07-07

    Genetic technologies based on transposon-mediated transgenesis along with several recently developed genome-editing technologies have become the preferred methods of choice for genetically manipulating many organisms. The silkworm, Bombyx mori, is a Lepidopteran insect of great economic importance because of its use in silk production and because it is a valuable model insect that has greatly enhanced our understanding of the biology of insects, including many agricultural pests. In the past 10 years, great advances have been achieved in the development of genetic technologies in B. mori, including transposon-based technologies that rely on piggyBac-mediated transgenesis and genome-editing technologies that rely on protein- or RNA-guided modification of chromosomes. The successful development and application of these technologies has not only facilitated a better understanding of B. mori and its use as a silk production system, but also provided valuable experiences that have contributed to the development of similar technologies in non-model insects. This review summarizes the technologies currently available for use in B. mori, their application to the study of gene function and their use in genetically modifying B. mori for biotechnology applications. The challenges, solutions and future prospects associated with the development and application of genetic technologies in B. mori are also discussed.

  10. Transcriptome Analysis of Silkworm, Bombyx mori, during Early Response to Beauveria bassiana Challenges

    PubMed Central

    Hou, Chengxiang; Qin, Guangxing; Liu, Ting; Geng, Tao; Gao, Kun; Pan, Zhonghua; Qian, Heying; Guo, Xijie

    2014-01-01

    Host–pathogen interactions are complex processes and it is a central challenge to reveal these interactions. Fungal infection of silkworm, Bombyx mori, may induce a variety of responsive reaction. However, little is known about the molecular mechanism of silkworm immune response against the fungal infection. To obtain an overview of the interaction between silkworm and an entomopathogenic fungus Beauveria bassiana, Digital Gene Expression profiling, a tag based high-throughput transcriptome sequencing method, was employed to screen and identify differentially expressed genes (DEGs, FDR≤0.001, ∣log2ratio∣≥1) of silkworm larvae during early response against B. bassiana infection. Total 1430 DEGs including 960 up-regulated and 470 down-regulated ones were identified, of which 627 DEGs can be classified into GO categories by Gene Ontology (GO) analysis. KEGG pathways analysis of these DEGs suggested that many biological processes, such as defense and response, signal transduction, phagocytosis, regulation of gene expression, RNA splicing, biosynthesis and metabolism, protein transport etc. were involved in the interaction between the silkworm and B. bassiana. A number of differentially expressed fungal genes were also identified by mapping the sequencing tags to B. bassiana genome. These results provided new insights to the molecular mechanism of silkworm immune response to B. bassiana infection. PMID:24618587

  11. Physicochemical Properties of Meat Batter Added with Edible Silkworm Pupae (Bombyx mori) and Transglutaminase

    PubMed Central

    Choi, Yun-Sang

    2017-01-01

    This study was conducted to investigate the physicochemical properties of meat batters prepared with fresh pork meat, back fat, water, and salt and formulated with three different amounts (5%, 10%, and 15%) of silkworm pupae (Bombyx mori) powder and transglutaminase (TG). Meat batters formulated with silkworm pupae powder showed significantly higher contents of protein and ash than control batter. Addition of silkworm pupae to batter also showed significantly lower cooking loss than the control. Moreover, meat batter containing 15% silkworm pupae showed no significant difference in redness value compared to the control. In addition, pH, viscosity, hardness, gumminess, and chewiness were improved after the addition of silkworm pupae. Furthermore, meat batter formulated with TG and silkworm pupae showed improved hardness, gumminess, chewiness and viscosity compared to control batter. Addition of 1% TG with 15% silkworm pupae to meat batter resulted in significantly higher pH, textures, and viscosity. Our data suggest that both silkworm pupae and TG can be added to meat batter to improve its physicochemical properties. Therefore, combination of silkworm pupae and TG could be a new nutritional and functional source for meat products. PMID:28747820

  12. Physicochemical Properties of Meat Batter Added with Edible Silkworm Pupae (Bombyx mori) and Transglutaminase.

    PubMed

    Park, Yoo-Sun; Choi, Yun-Sang; Hwang, Ko-Eun; Kim, Tae-Kyung; Lee, Cheol-Won; Shin, Dong-Min; Han, Sung Gu

    2017-01-01

    This study was conducted to investigate the physicochemical properties of meat batters prepared with fresh pork meat, back fat, water, and salt and formulated with three different amounts (5%, 10%, and 15%) of silkworm pupae (Bombyx mori) powder and transglutaminase (TG). Meat batters formulated with silkworm pupae powder showed significantly higher contents of protein and ash than control batter. Addition of silkworm pupae to batter also showed significantly lower cooking loss than the control. Moreover, meat batter containing 15% silkworm pupae showed no significant difference in redness value compared to the control. In addition, pH, viscosity, hardness, gumminess, and chewiness were improved after the addition of silkworm pupae. Furthermore, meat batter formulated with TG and silkworm pupae showed improved hardness, gumminess, chewiness and viscosity compared to control batter. Addition of 1% TG with 15% silkworm pupae to meat batter resulted in significantly higher pH, textures, and viscosity. Our data suggest that both silkworm pupae and TG can be added to meat batter to improve its physicochemical properties. Therefore, combination of silkworm pupae and TG could be a new nutritional and functional source for meat products.

  13. Involvement of Bombyx mori nucleopolyhedrovirus ORF41 (Bm41) in BV production and ODV envelopment.

    PubMed

    Tian, Cai-Hong; Zhao, Jin-Fang; Xu, Yi-Peng; Xue, Jian; Zhang, Bao-Qin; Cui, Ying-Jun; Zhang, Min-Juan; Bao, Yan-Yuan; Zhang, Chuan-Xi

    2009-04-25

    Bombyx mori nucleopolyhedrovirus (BmNPV) ORF41 (Bm41), homologous to Ac52, is a gene present in most lepidopteran nucleopolyhedroviruses. Bm41 transcripts and encoded protein in BmNPV-infected cells can be detected from 3 and 6 h post-infection, respectively. Immunoassays have shown that Bm41 is not a viral structural protein and is detected in both the nuclei and cytoplasm of infected cells. A Bm41-disrupted virus (vBm(De)) and a repaired virus (vBm(Re)) were generated to investigate the function of Bm41. The results showed that Bm41 was essential for viral replication, and the disruption of Bm41 resulted in a much lower viral titer. Transmission electron microscopy revealed that disruption of Bm41 affected normal nucleocapsid envelopment and polyhedra formation in the nucleus. The disruption of Bm41 might severely affect odv-ec27 and polyhedrin expression. The disrupted virus reduced BmNPV infectivity in an LD(50) bioassay and took 18-23 h longer to kill larvae than wild-type virus in an LT(50) bioassay.

  14. Editing of the heavy chain gene of Bombyx mori using transcription activator like effector nucleases.

    PubMed

    Wang, Yujun; Nakagaki, Masao

    2014-07-18

    The silk gland of Bombyx mori represents an established in vivo system for producing recombinant proteins. However, low yields of recombinant proteins have limited the system's further development because endogenous silk proteins were present. Transcription activator-like effector nucleases (TALENs) tool which work in pairs to bind and cleave DNA at specific sites, have recently been shown to be effective for genome editing in various organisms, including silkworms. To improve the yield of recombinant proteins synthesized in the silkworm by eliminated competition with endogenous fibroin synthesis, the heavy chain (H-chain) gene was knocked out using transcription activator-like effector nucleases (TALENs). A pair of TALENs that targets the 1st exon in the H-chain gene was synthesized and microinjected into silkworm embryos; the injected silkworms were screened for H-chain gene knock out (H-KO) based on their sericin cocoon-making characteristics. Sequence analysis revealed that the H-chain of the mutation was successfully edited. The TALENs was very efficient in editing the genome DNA of silkworm. By being eliminated competition with the H-chain, the production of recombinant proteins would be expected to increase markedly if this H-KO system is used.

  15. Characterization and recombinant protein expression of ferritin light chain homologue in the silkworm, Bombyx mori.

    PubMed

    Hong, Sun Mee; Mon, Hiroaki; Lee, Jae Man; Kusakabe, Takahiro

    2014-04-01

    The silkworm genome encodes three iron storage proteins or ferritins, Fer1HCH, Fer2LCH, and Fer3HCH. Probing our EST library constructed from 1-day-old silkworm eggs revealed only Fer2LCH mRNA, which encoded for a protein with a predicted putative N-glycosylation site. Developmental and tissue expression analyses during embryogenesis revealed that Fer2LCH mRNA was abundant from 6 h to 6 days after oviposition. Transcriptional expression of Fer2LCH during the postembryonic stage is also high in the larval fat body and mid-gut, and then is upregulated in all pupal tissues tested. We found that Fer2LCH mRNA contains an iron-responsive element, suggesting this ferritin subunit is subject to translational control. Although ferritin expression has been shown to increase following immune challenge in other insects, the levels of Fer2LCH mRNA were not significantly induced following viral or bacterial infection of Bombyx mori. Using a baculovirus expression system we expressed recombinant BmFer2LCH protein, which was detectable in the cytoplasmic fraction, likely in a compartment of the secretory pathway, and was shown to undergo posttranslational modifications including N-glycosylation. In particular, rBmFer2LCH carbohydrate chains were composed of mannose and GlcNAc. We suggest that Fer2LCH is important for iron homeostasis and maintaining normal organ function in silkworms.

  16. Silkworm (Bombyx mori) hemolymph unable to substitute fetal bovine serum in insect cell culture

    NASA Astrophysics Data System (ADS)

    Suparto, Irma H.; Khalam, Chandra Nur; Praira, Willy; Sajuthi, Dondin

    2014-03-01

    Fetal Bovine Serum (FBS) in animal cell culture media is an important source of nutrients for cell growth. However, the harvest and collection of FBS cause bioethical concerns. Efforts to reduce and preferably replace FBS with synthetic or other natural alternatives are continually being explored. Hemolymph silkworm (Bombyx mori) contains many nutrients needed for the process of metamorphosis. Therefore, there is possibility as an alternative nutritional supplement for cell culture to reduce the use of FBS. The objective of this study was to evaluate the macrocomponent of hemolymph and the possibility as medium supplement for Spodoptera fugiperda (Sf9) cell culture. Proximate analyses showed that hemolymph contains 89.76% of water, 2.52 mg/mL carbohydrate, 2.35% fat and 55.61 mg/mL protein. Further protein analysis, it consists of 15 fractions containing molecular weight of 22 - 152 kDa. The use of hemolymph as FBS substitution in Sf9 cell culture with various concentrations was unable to maintain and support cell growth. Further research still needed by prior adaptation of the tissue culture to minimal nutrition media before introduction of the hemolymph as supplement.

  17. Simple sequence repeat-based consensus linkage map of Bombyx mori.

    PubMed

    Miao, Xue-Xia; Xub, Shi-Jie; Li, Ming-Hui; Li, Mu-Wang; Huang, Jian-Hua; Dai, Fang-Yin; Marino, Susan W; Mills, David R; Zeng, Peiyu; Mita, Kazuei; Jia, Shi-Hai; Zhang, Yong; Liu, Wen-Bin; Xiang, Hui; Guo, Qiu-Hong; Xu, An-Ying; Kong, Xiang-Yin; Lin, Hong-Xuan; Shi, Yao-Zhou; Lu, Gang; Zhang, Xianglin; Huang, Wei; Yasukochi, Yuji; Sugasaki, Toshiyuki; Shimada, Toru; Nagaraju, Javaregowda; Xiang, Zhong-Huai; Wang, Sheng-Yue; Goldsmith, Marian R; Lu, Cheng; Zhao, Guo-Ping; Huang, Yong-Ping

    2005-11-08

    We established a genetic linkage map employing 518 simple sequence repeat (SSR, or microsatellite) markers for Bombyx mori (silkworm), the economically and culturally important lepidopteran insect, as part of an international genomics program. A survey of six representative silkworm strains using 2,500 (CA)n- and (CT)n-based SSR markers revealed 17-24% polymorphism, indicating a high degree of homozygosity resulting from a long history of inbreeding. Twenty-nine SSR linkage groups were established in well characterized Dazao and C108 strains based on genotyping of 189 backcross progeny derived from an F(1) male mated with a C108 female. The clustering was further focused to 28 groups by genotyping 22 backcross progeny derived from an F(1) female mated with a C108 male. This set of SSR linkage groups was further assigned to the 28 chromosomes (established linkage groups) of silkworm aided by visible mutations and cleaved amplified polymorphic sequence markers developed from previously mapped genes, cDNA sequences, and cloned random amplified polymorphic DNAs. By integrating a visible mutation p (plain, larval marking) and 29 well conserved genes of insects onto this SSR-based linkage map, a second generation consensus silkworm genetic map with a range of 7-40 markers per linkage group and a total map length of approximately 3431.9 cM was constructed and its high efficiency for genotyping and potential application for synteny studies of Lepidoptera and other insects was demonstrated.

  18. Enhancement of antioxidant ability of Bombyx mori silk fibroins by enzymatic coupling of catechin.

    PubMed

    Qi, Chenglong; Wang, Ping; Cui, Li; Deng, Chao; Yu, Yuanyuan; Wang, Qiang; Fan, Xuerong

    2016-02-01

    Enzymatic modification of Bombyx mori silk fibroin was carried out by using (+)-catechin, aiming at improving the antioxidant ability of the fibroin-based materials.The actions of tyrosinase on catechin were evaluated by using spectrophotometry, LC-MS, and Fourier transform infrared spectroscopy (FTIR). ε-Polylysine (ε-PLL) was used to investigate the possibility of the covalent reaction between catechin and the primary amine compound. The properties of the fibroin membranes before and after grafting of catechin were compared. The results revealed that catechin was oxidized into reactive o-quinones and subsequently formed catechin derivatives.Meanwhile, catechin could be efficiently grafted onto ε-PLL and led to a decrease in the amount of primary amine groups. 1H-NMR analysis verified the occurrence of the tyrosinase-catalyzed coupling of catechin onto the surface of silk fibroins. Improved antioxidant activity and better durability were obtained for the silk fibroin membrane based on catechin/tyrosinase treatment. Thermal behavior and biocompatibility for the catechin-grafted fibroin membranes did not noticeably change as compared to that of the untreated sample. The present work provided a novel method for preparation of the fibroin-based materials for biomedical applications.

  19. Transcriptional Response of Silkworm (Bombyx mori) Eggs to O2 or HCl Treatment

    PubMed Central

    Gong, Jing; Tian, Sha; Zhou, Xia; Yang, Huan; Zhu, Yong; Hou, Yong

    2016-01-01

    Diapause is a common biological phenomenon that occurs in many organisms, including fish, insects, and nematodes. In the silkworm (Bombyx mori), diapause generally occurs in the egg stage. Treatment with O2, HCl, or other compounds can prevent egg diapause. Here, we characterized the transcriptomic responses of newly laid eggs treated with O2 or HCl. Digital gene expression analysis showed that 610 genes in O2-treated eggs and 656 in HCl-treated eggs were differentially expressed. Of these, 343 genes were differentially expressed in both treatments. In addition to trehalases, sorbic acid dehydrogenases, and some enzymes involved in the carbohydrate metabolism, we also identified heat shock proteins, cytochrome P450, and GADD45, which are related to stress tolerance. Gene ontology enrichment analysis showed differentially expressed genes in O2-treated eggs were involved in oxidoreductase activity as well as in binding, catalytic, and metabolic processes. The Kyoto Encyclopedia of Genes and Genomes analysis showed that the pathways for ribosome biogenesis, spliceosome, and circadian rhythm were significantly enriched in HCl-treated eggs. The reliability of the data was confirmed by qRT-PCR analysis. Our results improved the understanding of the mechanism of diapause blocking in silkworm eggs treated with O2 or HCl and identified novel molecular targets for future studies. PMID:27941611

  20. siRNAs induce efficient RNAi response in Bombyx mori embryos.

    PubMed

    Yamaguchi, Junichi; Mizoguchi, Takayuki; Fujiwara, Haruhiko

    2011-01-01

    Short interference RNA (siRNA) is widely used in mammalian cells. In insects, however, reports concerning the suitablility of siRNA in vivo is very limited compared with that of long dsRNA, which is thought to be more effective. There is insufficient information on the essential rules of siRNA design in insects, as very few siRNAs have been tested in this context. To establish an effective method of gene silencing using siRNA in vivo in insects, we determined the effects of siRNA on seven target genes. We designed siRNAs according to a new guideline and injected them into eggs of Bombyx mori. At the mRNA level, the expression of most of these genes was successfully silenced, down to less than half the constitutive level, which in some cases led to the development of distinctive phenotypes. In addition, we observed stronger effect of siRNA both on the mRNA level and the phenotype than that of long dsRNA under comparable conditions. These results indicate that direct injection of siRNA is an effective reverse-genetics tool for the analysis of embryogenesis in vivo in insects.

  1. Identification of the key stages for sex determination in the silkworm, Bombyx mori.

    PubMed

    Sakai, Hiroki; Aoki, Fugaku; Suzuki, Masataka G

    2014-03-01

    In general, the master switch gene for sex determination is expressed for a limited period during the early embryonic stage. To increase our understanding of the sex determination mechanism in Bombyx mori, it is important to understand when sex determination takes place. To examine the key stages for sex determination in this insect, we focused on the expression patterns of Bmdsx (a double-switch gene in the sex determination cascade of B. mori) and BmIMP (a gene expressed specifically in males involved in male-specific splicing of Bmdsx). Reverse transcription PCR (RT-PCR) analysis revealed that male-type Bmdsx expression was observed in females at 27 and 29 h after oviposition (hao), and finally disappeared at 32 hao. Moreover, BmIMP mRNA was also expressed in these females, and its expression level was comparable to that of the male-type Bmdsx mRNA. These results demonstrated that female embryos before 32 hao can show male-type expression of Bmdsx and BmIMP, suggesting that sex determination occurs between 29 and 32 hao, which correspond to the developmental stages from the head lobe differentiation to spoon-shaped embryo stages. This also suggests that the master switch gene for sex determination of B. mori is expressed in females during this period and represses the male-specific mode of expression in sex-determining genes.

  2. Bombyx mori Serpin6 regulates prophenoloxidase activity and the expression of antimicrobial proteins.

    PubMed

    Li, Bing; Yu, Hai-Zhong; Ye, Chong-Jun; Ma, Yan; Li, Xing; Fan, Tao; Chen, Fu-Sheng; Xu, Jia-Ping

    2017-04-30

    Serpins are a family of serine protease inhibitors that are found widely in insects. They play an important role in insect physiological responses, such as innate immunity and development. In this study, we obtained the Bombyx mori serpin6 (BmSerpin6) sequence from National Center for Biotechnology Information (NCBI) and the silkworm genome database (SilkDB). Reverse transcription PCR (RT-PCR) results showed that BmSerpin6 was expressed highly in hemocytes, the midgut, and the fat body. After challenging with Micrococcus luteus (Mi) and Serratia marcescens (Sm), the BmSerpin6 expression level was induced significantly. Transcript levels of gloverin2 and prophenoloxidase (PPO) activity were reduced significantly in the fat body and hemocytes after injecting the recombinant BmSerpin6 protein into silkworm larvae. A BmSerpin6 recombinant plasmid (BmSerpin6-pAC 5.1) was constructed successfully and transfected into Drosophila S2 cells, which resulted in significantly reduced expression of the drosomycin protein. These results indicated that BmSerpin6 might regulate silkworm immune responses. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Enhancement of larval RNAi efficiency by over-expressing Argonaute2 in Bombyx mori.

    PubMed

    Li, Zhiqian; Zeng, Baosheng; Ling, Lin; Xu, Jun; You, Lang; Aslam, Abu F M; Tan, Anjiang; Huang, Yongping

    2015-01-01

    RNA interference has been described as a powerful genetic tool for gene functional analysis and a promising approach for pest management. However, RNAi efficiency varies significantly among insect species due to distinct RNAi machineries. Lepidopteran insects include a large number of pests as well as model insects, such as the silkworm, Bombyx mori. However, only limited success of in vivo RNAi has been reported in lepidoptera, particularly during the larval stages when the worms feed the most and do the most harm to the host plant. Enhancing the efficiency of larval RNAi in lepidoptera is urgently needed to develop RNAi-based pest management strategies. In the present study, we investigate the function of the conserved RNAi core factor, Argonaute2 (Ago2), in mediating B. mori RNAi efficiency. We demonstrate that introducing BmAgo2 dsRNA inhibits the RNAi response in both BmN cells and embryos. Furthermore, we establish several transgenic silkworm lines to assess the roles of BmAgo2 in larval RNAi. Over-expressing BmAgo2 significantly facilitated both dsRNA-mediated larval RNAi when targeting DsRed using dsRNA injection and shRNA-mediated larval RNAi when targeting BmBlos2 using transgenic shRNA expression. Our results show that BmAgo2 is involved in RNAi in B. mori and provides a promising approach for improving larval RNAi efficiency in B. mori and in lepidopteran insects in general.

  4. Immobilization of foreign protein into polyhedra of Bombyx mori nucleopolyhedrovirus (BmNPV)*

    PubMed Central

    Xiang, Xing-wei; Yang, Rui; Chen, Lin; Hu, Xiao-long; Yu, Shao-fang; Cao, Cui-ping; Wu, Xiao-feng

    2012-01-01

    In the late phase of Bombyx mori nucleopolyhedrovirus (BmNPV) infection, a large amount of polyhedra appear in the infected cell nucleolus, these polyhedra being dense protein crystals protecting the incorporated virions from the harsh environment. To investigate whether the foreign protein could be immobilized into the polyhedra of BmNPV, two recombinant baculoviruses were generated by a novel BmNPV polyhedrin-plus (polh+) Bac-to-Bac system, designated as vBmBac(polh+)-enhanced green fluorescent protein (EGFP) and vBmBac(polh+)-LacZ, which can express the polyhedrin and foreign protein simultaneously. Light microscopy analysis showed that all viruses produced polyhedra of normal appearance. Green fluorescence can be apparently detected on the surface of the vBmBac(polh+)-EGFP polyhedra, but not the BmNPV polyhedra. Fluorescence analysis and anti-desiccation testing confirmed that EGFP was embedded in the polyhedra. As expected, the vBmBac(polh+)-LacZ polyhedra contained an amount of LacZ and had a higher β-galactosidase activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were also performed to verify if the foreign proteins were immobilized into polyhedra. This study provides a new inspiration for efficient preservation of useful proteins and development of new pesticides with toxic proteins. PMID:22302424

  5. Female qualities in males: vitellogenin synthesis induced by ovary transplants into the male silkworm, Bombyx mori.

    PubMed

    Yang, Congwen; Lin, Ying; Shen, Guanwang; Chen, Enxiang; Wang, Yanxia; Luo, Juan; Zhang, Haiyan; Xing, Runmiao; Xia, Qingyou

    2014-10-10

    Female qualities in males are common in vertebrates but have not been extensively reported in insects. Vitellogenin (Vg) is highly expressed in the female fat body and is generally required for the formation of yolk proteins in the insect egg. Vg upregulation is generally regarded as a female quality in female oviparous animals. In this study, we found that Bombyx mori Vg (BmVg) is especially highly expressed in the female pupa. Downregulation of the BmVg gene in the female pupa by RNA interference (RNAi) interfered with egg formation and embryonic development, showing the importance of BmVg in these processes. So, we used BmVg as a biomarker for female qualities in the silkworm. Hematoxylin-eosin staining and immunofluorescence histochemistry showed that ovary transplants induced BmVg synthesis in the male pupa fat body. Ovaries transplanted into male silkworms produced only a few eggs with deformed yolk granules. These results suggested that the amount of BmVg in the male silkworm was insufficient for eggs to undergo complete embryonic development. After 17-beta-estradiol was used to treat male pupae and male pupal fat bodies, BmVg was upregulated in vivo and in vitro. These findings indicated that the male silkworm has innate female qualities that were induced by a transplanted ovary and 17β-estradiol. However, in silkworms, female qualities in males are not as complete as in females.

  6. Aspartate Decarboxylase is Required for a Normal Pupa Pigmentation Pattern in the Silkworm, Bombyx mori.

    PubMed

    Dai, Fangyin; Qiao, Liang; Cao, Cun; Liu, Xiaofan; Tong, Xiaoling; He, Songzhen; Hu, Hai; Zhang, Li; Wu, Songyuan; Tan, Duan; Xiang, Zhonghuai; Lu, Cheng

    2015-06-16

    The pigmentation pattern of Lepidoptera varies greatly in different development stages. To date, the effects of key genes in the melanin metabolism pathway on larval and adult body color are distinct, yet the effects on pupal pigmentation remains unclear. In the silkworm, Bombyx mori, the black pupa (bp) mutant is only specifically melanized at the pupal stage. Using positional cloning, we found that a mutation in the Aspartate decarboxylase gene (BmADC) is causative in the bp mutant. In the bp mutant, a SINE-like transposon with a length of 493 bp was detected ~2.2 kb upstream of the transcriptional start site of BmADC. This insertion causes a sharp reduction in BmADC transcript levels in bp mutants, leading to deficiency of β-alanine and N-β-alanyl dopamine (NBAD), but accumulation of dopamine. Following injection of β-alanine into bp mutants, the color pattern was reverted that of the wild-type silkworms. Additionally, melanic pupae resulting from knock-down of BmADC in the wild-type strain were obtained. These findings show that BmADC plays a crucial role in melanin metabolism and in the pigmentation pattern of the silkworm pupal stage. Finally, this study contributes to a better understanding of pupa pigmentation patterns in Lepidoptera.

  7. Role of BmDredd during Apoptosis of Silk Gland in Silkworm, Bombyx mori

    PubMed Central

    Chen, Rui-ting; Jiao, Peng; Liu, Zhen; Lu, Yan; Xin, Hu-hu; Zhang, Deng-pan; Miao, Yun-gen

    2017-01-01

    Silk glands (SGs) undergo massive apoptosis driven degeneration during the larval-pupal transformation. To better understand this event on molecular level, we investigated the expression of apoptosis-related genes across the developmental transition period that spans day 4 in the fifth instar Bombyx mori larvae to day 2 pupae. Increases in the expression of BmDredd (an initiator caspase homolog) closely followed the highest BmEcR expression and resembled the expression trend of BmIcE. Simultaneously, we found that BmDredd expression was significantly higher in SG compared to other tissues at 18 h post-spinning, but reduced following injection of the apoptosis inhibitor (Z-DEVD-fmk). Furthermore, BmDredd expression correlated with changes of caspase3-like activities in SG and RNAi-mediated knockdown of BmDredd delayed SG apoptosis. Moreover, caspase3-like activity was increased in SG by overexpression of BmDredd. Taken together, the results suggest that BmDredd plays a critical role in SG apoptosis. PMID:28068357

  8. Rectal sac distention is induced by 20-hydroxyecdysone in the pupa of Bombyx mori.

    PubMed

    Suzuki, Takumi; Sakurai, Sho; Iwami, Masafumi

    2009-03-01

    Holometabolous insects do not excrete but store metabolic wastes during the pupal period. The waste is called meconium and is purged after adult emergence. Although the contents of meconium are well-studied, the developmental and physiological regulation of meconium accumulation is poorly understood. In Bombyx mori, meconium is accumulated in the rectal sac; thereby, the rectal sac distends at the late pupal stage. Here, we show that rectal sac distention occurs between 4 and 5 days after pupation. The distention is halted by brain-removal just after larval-pupal ecdysis but not by brain-removal 1 day after pupation. In the pupae, brain-removal just after ecdysis kept the hemolymph ecdysteroid titer low during early and mid-pupal stages. An injection of 20-hydroxyecdysone (20E) evoked the distention that was halted by brain-removal in a dose-dependent manner. Therefore, brain-removal caused the lack of ecdysteroid, and rectal sac distention did not appear in the brain-removed pupae because of the lack of ecdysteroid. We conclude that rectal sac distention is one of the developmental events regulated by 20E during the pupal period in B. mori.

  9. Mechanical properties of cocoons constructed consecutively by a single silkworm caterpillar, Bombyx mori

    NASA Astrophysics Data System (ADS)

    Huang, S. Q.; Zhao, H. P.; Feng, X. Q.; Cui, W.; Lin, Z.; Xu, M. Q.

    2008-04-01

    Most animals have the ability to adapt, to some extends and in different ways, the variation or disturbance of environment. In our experiments, we forced a silkworm caterpillar to spin two, three or four thin cocoons by taking it out from the cocoon being constructed. The mechanical properties of these cocoons were studied by static tensile tests and dynamic mechanical thermal analysis. Though external disturbances may cause the decrease in the total weight of silk spun by the silkworm, a gradual enhancement was interestingly found in the mechanical properties of these thin cocoons. Scanning electron microscopy observations of the fractured specimens of the cocoons showed that there exist several different energy dissipation mechanisms occurred simultaneously at macro-, meso-, and micro-scales, yielding a superior capacity of cocoons to adsorb the energy of possible attacks from the outside and to protect efficiently its pupa against damage. Through evolution of millions of years, therefore, the silkworm Bombyx mori seems to have gained the ability to adapt external disturbances and to redesign a new cocoon with optimized protective function when its first cocoon has been damaged for some reasons.

  10. Production of Bombyx mori silk fibroin incorporated with unnatural amino acids.

    PubMed

    Teramoto, Hidetoshi; Kojima, Katsura

    2014-07-14

    Silk fibroin incorporated with unnatural amino acids was produced by in vivo feeding of p-chloro-, p-bromo-, and p-azido-substituted analogues of L-phenylalanine (Phe) to transgenic silkworms (Bombyx mori) that expressed a mutant of phenylalanyl-tRNA synthetase with expanded substrate recognition capabilities in silk glands. Cutting down the content of Phe in the diet was effective for increasing the incorporation of Phe analogues but simultaneously caused a decrease of fibroin production. The azide groups incorporated in fibroin were active as chemical handles for click chemistry in both the solubilized and the solid (fibrous) states. The azides survived degumming in the boiling alkaline solution that is required for complete removal of the sericin layer, demonstrating that AzPhe-incorporated silk fibroin could be a versatile platform to produce "clickable" silk materials in various forms. This study indicates the huge potential of UAA mutagenesis as a novel methodology to alter the characteristics of B. mori silk.

  11. Differentially expressed genes in the fat body of Bombyx mori in response to phoxim insecticide.

    PubMed

    Gu, Z Y; Li, F C; Wang, B B; Xu, K Z; Ni, M; Zhang, H; Shen, W D; Li, B

    2015-01-01

    The silkworm, Bombyx mori, is an economically important insect. However, poisoning of silkworms by organophosphate pesticides causes tremendous loss to the sericulture. The fat body is the major tissue involved in detoxification and produces antimicrobial peptides and regulates hormones. In this study, a microarray system comprising 22,987 oligonucluotide 70-mer probes was employed to examine differentially expressed genes in the fat body of B. mori exposed to phoxim insecticide. The results showed that a total of 774 genes were differentially expressed upon phoxim exposure, including 500 up-regulated genes and 274 down-regulated genes. The expression levels of eight detoxification-related genes were up-regulated upon phoxim exposure, including six cytochrome P450s and two glutathione-S-transferases. It was firstly found that eight antimicrobial peptide genes were down-regulated, which might provide important references for studying the larvae of B. mori become more susceptible to microbial infections after phoxim treatment. In addition, we firstly detected the expression level of metamorphosis-related genes after phoxim exposure, which may lead to impacted reproduction. Our results may facilitate the overall understanding of the molecular mechanism of multiple pathways following exposure to phoxim insecticide in the fat body of B. mori. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. A novel protease inhibitor in Bombyx mori is involved in defense against Beauveria bassiana.

    PubMed

    Li, Youshan; Zhao, Ping; Liu, Shiping; Dong, Zhaoming; Chen, Jianping; Xiang, Zhonghuai; Xia, Qingyou

    2012-10-01

    Entomopathogenic fungi, such as Beauveria bassiana, penetrate the insect cuticle using a plethora of hydrolytic enzymes including cuticle-degrading proteases and chitinases, which are important virulence factors. The insect integument and hemolymph contains a relatively high concentration of protease inhibitors, which are closely involved with defense against pathogenic microorganisms. To elucidate the molecular mechanism underlying resistance against entomopathogenic fungi and to identify a new molecular target for improving fungal resistance in the silkworm, Bombyx mori, we cloned and expressed a novel silkworm TIL-type protease inhibitor BmSPI38, which was very stable over a wide range of temperatures and pH values. An activity assay suggested that BmSPI38 potently inactivated the insecticidal cuticle-degrading enzyme (CDEP-1) produced by B. bassiana and subtilisin A produced by Bacillus licheniformis. The melanization of silkworm induced by CDEP-1 protease could also be blocked by BmSPI38. These results provided new insights into the molecular mechanisms whereby insect protease inhibitors provide resistance against entomopathogenic fungi, suggesting the possibility of using fungal biopesticides in sericulture. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Metabolomics differences between silkworms (Bombyx mori) reared on fresh mulberry (Morus) leaves or artificial diets.

    PubMed

    Dong, Hui-Ling; Zhang, Sheng-Xiang; Tao, Hui; Chen, Zhuo-Hua; Li, Xue; Qiu, Jian-Feng; Cui, Wen-Zhao; Sima, Yang-Hu; Cui, Wei-Zheng; Xu, Shi-Qing

    2017-09-08

    Silkworms (Bombyx mori) reared on artificial diets have great potential applications in sericulture. However, the mechanisms underlying the enhancement of metabolic utilization by altering silkworm nutrition are unclear. The aim of this study was to investigate the mechanisms responsible for the poor development and low silk protein synthesis efficiency of silkworms fed artificial diets. After multi-generational selection of the ingestive behavior of silkworms to artificial diets, we obtained two strains, one of which developed well and another in which almost all its larvae starved to death on the artificial diets. Subsequently, we analyzed the metabolomics of larval hemolymph by gas chromatography/liquid chromatography-mass spectrometry, and the results showed that vitamins were in critically short supply, whereas the nitrogen metabolic end product of urea and uric acid were enriched substantially, in the hemolymph of the silkworms reared on the artificial diets. Meanwhile, amino acid metabolic disorders, as well as downregulation of carbohydrate metabolism, energy metabolism, and lipid metabolism, co-occurred. Furthermore, 10 male-dominant metabolites and 27 diet-related metabolites that differed between male and female silkworms were identified. These findings provide important insights into the regulation of silkworm metabolism and silk protein synthesis when silkworms adapt to an artificial diet.

  14. Knockdown of Broad-Complex Gene Expression of Bombyx mori by Oligopyrrole Carboxamides Enhances Silk Production.

    PubMed

    Ali, Asfa; Bovilla, Venugopal Reddy; Mysarla, Danti Kumari; Siripurapu, Prasanthi; Pathak, Rashmi U; Basu, Bhakti; Mamillapalli, Anitha; Bhattacharya, Santanu

    2017-04-11

    Bombyx mori (B. mori) is important due to its major role in the silk production. Though DNA binding ligands often influence gene expression, no attempt has been made to exploit their use in sericulture. The telomeric heterochromatin of B. mori is enriched with 5'-TTAGG-3' sequences. These sequences were also found to be present in several genes in the euchromatic regions. We examined three synthetic oligopyrrole carboxamides that target 5'-TTAGG-3' sequences in controlling the gene expression in B. mori. The ligands did not show any defect or feeding difference in the larval stage, crucial for silk production. The ligands caused silencing of various isoforms of the broad-complex transcription factor and cuticle proteins which resulted in late pupal developmental defects. Furthermore, treatment with such drugs resulted in statistically enhanced cocoon weight, shell weight, and silk yield. This study shows for the first time use of oligopyrrole carboxamide drugs in controlling gene expression in B. mori and their long term use in enhancing silk production.

  15. Differentially expressed microRNAs in diapausing versus HCl-treated Bombyx embryos

    PubMed Central

    Qin, Mingyue; Lin, Bimin; Chen, Fangyan; Yan, Huichao; Li, Wenchu

    2017-01-01

    Differentially expressed microRNAs were detected to explore the molecular mechanisms of diapause termination. The total small RNA of diapause-destined silkworm eggs and HCl-treated eggs was extracted and then sequenced using HiSeq high-throughput method. 44 novel miRNAs were discovered. Compared to those in the diapause-destined eggs, 61 miRNAs showed significant changes in the acid-treated eggs, with 23 being up-regulated and 38 being down-regulated. The potential target genes of differentially expressed miRNAs were predicted by miRanda. Gene Ontology and KEGG pathway enrichment analysis of these potential target genes revealed that they were mainly located within cells and organelles, involved in cellular and metabolic processes, and participated in protein production, processing and transportation. Two differentially expressed genes, Bombyx mori SDH and Bmo-miR-2761-3p, were further analyzed with qRT-PCR. BmSDH was significantly up-regulated in the HCl-treated eggs, while Bmo-miR-2761-3p was down-regulated. These results suggested that these two genes were well coordinated in silkworm eggs. Dual luciferase reporter assay demonstrated that Bmo-miR-2761-3p inhibited the expression of BmSDH. PMID:28700597

  16. Functional Loss of Bmsei Causes Thermosensitive Epilepsy in Contractile Mutant Silkworm, Bombyx mori

    NASA Astrophysics Data System (ADS)

    Nie, Hongyi; Cheng, Tingcai; Huang, Xiaofeng; Zhou, Mengting; Zhang, Yinxia; Dai, Fangyin; Mita, Kazuei; Xia, Qingyou; Liu, Chun

    2015-07-01

    The thermoprotective mechanisms of insects remain largely unknown. We reported the Bombyx mori contractile (cot) behavioral mutant with thermo-sensitive seizures phenotype. At elevated temperatures, the cot mutant exhibit seizures associated with strong contractions, rolling, vomiting, and a temporary lack of movement. We narrowed a region containing cot to ~268 kb by positional cloning and identified the mutant gene as Bmsei which encoded a potassium channel protein. Bmsei was present in both the cell membrane and cytoplasm in wild-type ganglia but faint in cot. Furthermore, Bmsei was markedly decreased upon high temperature treatment in cot mutant. With the RNAi method and injecting potassium channel blockers, the wild type silkworm was induced the cot phenotype. These results demonstrated that Bmsei was responsible for the cot mutant phenotype and played an important role in thermoprotection in silkworm. Meanwhile, comparative proteomic approach was used to investigate the proteomic differences. The results showed that the protein of Hsp-1 and Tn1 were significantly decreased and increased on protein level in cot mutant after thermo-stimulus, respectively. Our data provide insights into the mechanism of thermoprotection in insect. As cot phenotype closely resembles human epilepsy, cot might be a potential model for the mechanism of epilepsy in future.

  17. Molecular and neural mechanisms of sex pheromone reception and processing in the silkmoth Bombyx mori

    PubMed Central

    Sakurai, Takeshi; Namiki, Shigehiro; Kanzaki, Ryohei

    2014-01-01

    Male moths locate their mates using species-specific sex pheromones emitted by conspecific females. One striking feature of sex pheromone recognition in males is the high degree of specificity and sensitivity at all levels, from the primary sensory processes to behavior. The silkmoth Bombyx mori is an excellent model insect in which to decipher the underlying mechanisms of sex pheromone recognition due to its simple sex pheromone communication system, where a single pheromone component, bombykol, elicits the full sexual behavior of male moths. Various technical advancements that cover all levels of analysis from molecular to behavioral also allow the systematic analysis of pheromone recognition mechanisms. Sex pheromone signals are detected by pheromone receptors expressed in olfactory receptor neurons in the pheromone-sensitive sensilla trichodea on male antennae. The signals are transmitted to the first olfactory processing center, the antennal lobe (AL), and then are processed further in the higher centers (mushroom body and lateral protocerebrum) to elicit orientation behavior toward females. In recent years, significant progress has been made elucidating the molecular mechanisms underlying the detection of sex pheromones. In addition, extensive studies of the AL and higher centers have provided insights into the neural basis of pheromone processing in the silkmoth brain. This review describes these latest advances, and discusses what these advances have revealed about the mechanisms underlying the specific and sensitive recognition of sex pheromones in the silkmoth. PMID:24744736

  18. Identification of structural proteins of Bombyx mori parvo-like virus (China Zhenjiang isolate).

    PubMed

    Lv, Meng; Yao, Qin; Wang, Yong; Liu, Xiaoyong; Liu, Haijun; Huang, Guoping; Chen, Keping; Zhang, Junhong; Li, Xiaogang

    2011-01-01

    Bombyx mori parvo-like virus is a type of virus containing two single-stranded linear DNA molecules (VD1, VD2). In the present work, the structural proteins of B. mori parvo-like virus (China Zhenjiang isolate) (BmDNV-Z) were identified. The virions were purified by cesium chloride density gradient centrifugation from the feces of B. mori larvae infected with BmDNV-Z and analyzed by SDS-PAGE, Western blotting and MALDI-TOF/mass spectometry. There were seven polypeptides of the virions, named P1 to P7, which reacted with the anti-virion serum. In addition, two polypeptides, P5 and P6, were recognized by the antibody against a viral protein encoded by VD1-ORF3. The results showed that the viral polypeptides were mainly composed of 7 proteins named P1 to P7. Western blotting with anti-virion serum indicated that structural proteins P5, P6 and P7 had molecular weights of around 50, 55 and 130 kDa, respectively. They were the major immunogens of the virion. Among them, P5 and P6 were coded by VD1-ORF3 and P7 was encoded by VD2-ORF1. Taken together, three viral structural proteins encoded by VD1-ORF3 and VD2-ORF1 were identified in BmDNV-Z for the first time. Copyright © 2010 S. Karger AG, Basel.

  19. Vibrational infrared conformational studies of model peptides representing the semicrystalline domains of Bombyx mori silk fibroin.

    PubMed

    Taddei, Paola; Monti, Patrizia

    2005-08-05

    The structural organization of Bombyx mori silk fibroin was investigated by infrared (IR) spectroscopy. To this aim, (AG)15 and other model peptides of varying chain length, containing tyrosine (Y), valine (V), and serine (S) in the basic (AG)n sequence were synthesized by the solid phase method and their spectroscopic properties were determined. Both the position and the relative content of Y, V, and S residues in the (AG)n model system appeared critical in determining the preferred conformation, i.e., silk I, silk II, and unordered structures. Curve fitting analysis in the amide I range showed that the model peptides with prevailing silk II structure displayed different beta-sheet content, which was dependent on the degree of interruption of the (AG)n sequence. In this regard, the bands at about 1000 and 980 cm(-1), specifically assigned to the AG sequence of the B. mori silk fibroin chain, were identified as marker of the degree of interruption of the (AG)n sequence.A stable silk I structure was observed only when the Y residue was located near the chain terminus, while a silk I --> silk II conformational transition occurred when it was positioned in the central region of the peptide. Analysis of the second-derivative spectra in the amide I range allowed us to identify a band at 1639 cm(-1) (4 --> 1 hydrogen-bonded type II beta-turns), which is characteristic of the silk I conformation.

  20. Effects of Ag Nanoparticles on Growth and Fat Body Proteins in Silkworms (Bombyx mori).

    PubMed

    Meng, Xu; Abdlli, Nouara; Wang, Niannian; Lü, Peng; Nie, Zhichao; Dong, Xin; Lu, Shuang; Chen, Keping

    2017-03-30

    Ag nanoparticles (AgNPs), a widely used non-antibiotic, antibacterial material, have shown toxic and other potentially harmful effects in mammals. However, the deleterious effects of AgNPs on insects are still unknown. Here, we studied the effects of AgNPs on the model invertebrate organism Bombyx mori. After feeding silkworm larvae different concentrations of AgNPs, we evaluated the changes of B. mori body weights, survival rates, and proteomic differences. The results showed that low concentrations (<400 mg/L) of AgNPs promoted the growth and cocoon weights of B. mori. Although high concentrations (≥800 mg/L) of AgNPs also improved B. mori growth, they resulted in silkworm death. An analysis of fat body proteomic differences revealed 13 significant differences in fat body protein spots, nine of which exhibited significantly downregulated expression, while four showed significantly upregulated expression. Reverse transcription-polymerase chain reaction results showed that at an AgNP concentration of 1600 mg/L, the expression levels of seven proteins were similar to the transcription levels of their corresponding genes. Our results suggest that AgNPs lowered the resistance to oxidative stress, affected cell apoptosis, and induced cell necrosis by regulating related protein metabolism and metabolic pathways in B. mori.

  1. Comparative transcriptomic analysis of Bombyx mori fat body tissue following dietary restriction.

    PubMed

    Pan, Ye; Lü, Peng; Wang, Qinyun; Zhu, Feifei; Li, Chengjun; He, Yuanqing; Chen, Keping

    2017-05-01

    Dietary restriction (DR) refers to a reduction in food intake to induce undernutrition but not malnutrition, which extends the lifespan of multiple species. Although there are invertebrate aging models, such as the Caenorhabditis elegans and Drosophila melanogaster, aging studies in Lepidoptera are few in number and the underlying life-extending molecular mechanisms are not clear. Research on a broader range of animals is necessary to support generalizations on mechanisms of aging and rates of aging. The aim of this study was to further investigate genes and pathways associated with DR in Bombyx mori. Here, we used mRNA deep sequencing (RNA-seq) to further investigate genes and pathways associated with DR. The transcriptome profiles showed that most of the differentially expressed genes were upregulated following DR, and genes involved in amino acid and protein metabolism, RNA metabolism and translation, energy metabolism, nitrogen metabolism, and juvenile hormone pathway-related proteins were particularly affected. DR also affects the metabolism of uric acid and urea, which accumulated in silkworm following DR. We speculate that this may not be due to activation of uric acid biosynthesis, but rather by downregulating the degradation of uric acid and urea. These results may help us to understand the mechanisms by which DR prolong lifespan in insects and other animals. © 2017 Wiley Periodicals, Inc.

  2. Molecular characterization of a peritrophic membrane protein from the silkworm, Bombyx mori.

    PubMed

    Hu, Xiaolong; Chen, Lin; Yang, Rui; Xiang, Xingwei; Wu, Xiaofeng

    2013-02-01

    The peritrophic membrane lines the gut of most insects at one or more stages of their life cycles. It facilitates the digestive processes in the guts and protects from invasion by pathogens or food particles. In the current study, a novel PM protein, designated as BmMtch, was identified from the silkworm, Bombyx mori. The open reading frame of BmMtch is 888 bp in length, encoding 295 amino acid residues consisting of two domains (Mito_carr domains) and three transmembrane regions. They are localized on the 11th chromosome as single copy with one exon only. Quantitative real time PCR analysis (qRT-PCR) revealed that BmMtch was mainly expressed in larval fat bodies, Malpighian tubules, testis and ovaries, and could be detected through all stages of the life cycle of silkworm. Immuno-fluorescence analysis indicated that BmMtch was localized within the goblet cell of larval midgut. Western blotting analysis showed that BmMtch were detected in total proteins of PM and larval midgut. The characteristics of BmMtch indicated that BmMtch represents a novel member of insect PM proteins, without chitin-binding domains.

  3. An active recombinant cocoonase from the silkworm Bombyx mori: bleaching, degumming and sericin degrading activities.

    PubMed

    Unajak, Sasimanas; Aroonluke, Suradet; Promboon, Amornrat

    2015-04-01

    Cocoonase is a serine protease produced by silk moths and used for softening the cocoons so that they can escape. Degumming is one of the important steps in silk processing. This research aimed to produce an active recombinant Bombyx mori cocoonase (BmCoc) for the silk degumming process. A recombinant BmCoc was successfully expressed in a Pichia pastoris system. The purified enzyme showed specific activity of 227 U mg(-1) protein, 2.4-fold purification, 95% yield and a molecular weight of 26 kDa. The enzyme exhibited optimal temperature at 40 °C and optimal pH at 8, and showed thermal stability at 25-45 °C and pH stability at 5-9. The recombinant enzyme exhibited sericin degumming ability and color bleaching characteristics, and did not affect the fibroin fiber. The enzyme also degraded sericin substrate with a product size about 30-70 kDa. In this study, we successfully produced the active recombinant BmCoc in P. pastoris with promising functions for the Thai silk degumming process, which includes degumming, sericin degrading and color bleaching activities. Our data clearly indicated that the recombinant enzyme had proteolytic activity on sericin but not on fibroin proteins. The recombinant BmCoc has proven to be suitable for numerous applications in the silk industry. © 2014 Society of Chemical Industry.

  4. Centuries of domestication has not impaired oviposition site-selection function in the silkmoth, Bombyx mori.

    PubMed

    Damodaram, Kamala Jayanthi Pagadala; Kempraj, Vivek; Aurade, Ravindra Mahadappa; Rajasekhar, Sowmya Bandhisara; Venkataramanappa, Ravindra Kothapalli; Nandagopal, Bakthavatsalam; Verghese, Abraham

    2014-12-15

    Oviposition site-selection in insects is mediated through innate recognition templates (IRTs) tuned to specific chemical cues. These cues aid gravid insects in choosing suitable oviposition sites and may even enhance the fitness of their offspring by warding off predators and parasitoids. However, studies on the evolution of oviposition site-selection and cues instigating oviposition in domesticated insects remain elusive. Using the interaction between the silkmoth, Bombyx mori, and its host plant mulberry, Morus alba, as a model system, we demonstrate that centuries of domestication of silkmoth has not impaired its oviposition site-selection function. Silkmoths significantly preferred mulberry leaves to filter paper as oviposition sites. Oviposition assays with filter paper, filter paper treated with leaf volatiles and leaf alone proved that surface texture was not a significant criterion for oviposition site-selection, but volatile cues were. Oviposition assays with electrophysiologically active compounds from mulberry revealed that two of the volatiles, valencene and α-humulene, aided moths in choosing suitable oviposition sites and enhanced egg-laying significantly. Moreover, we show that generalist egg-parasitoids are strongly repelled by valencene and α-humulene. Our results demonstrate that IRTs tuned to cues that aid crucial functions like oviposition site-selection are less likely to be impaired even after centuries of domestication.

  5. Ultrastructural analysis of lipid incorporation in the embryonic silkworm, Bombyx mori.

    PubMed

    Yamahama, Yumi; Muranaka, Yoshinori; Kumakiri, Yoko; Tamotsu, Satoshi; Hariyama, Takahiko

    2009-05-01

    Insect eggs store many lipid droplets as an energy source for embryonic development. We previously reported that lipid droplets are incorporated into embryos in three steps in the silkworm, Bombyx mori. The midgut plays important roles in lipid incorporation during the second and third steps, whereas the manner of lipid incorporation during the first step is still unknown. In this study, we focused on how lipids were incorporated into the embryo in the first step, compared with the mechanisms used in the second step, by means of transmission electron microscopy using the high-pressure freezing and freeze substitution method. At the beginning of the first step (blastoderm formation stage), some lipid droplets were observed in each cell of the embryonic tissues. Lipid droplets were seen to be derived from the oocyte peripheral cytoplasm by superficial cleavage. At the end of the first step (late appendage formation stage), some lipid droplets were attached to the elongated rough endoplasmic reticulum (rER). It seemed that formation of the lipid droplets occurred in embryonic cells at the end of the first step, because the rER is the site of biogenesis of lipid droplets. The incorporation of lipid droplets in the first step may be subdivided into two stages: the blastoderm formation stage and the subsequent stage before blastokinesis.

  6. Transcriptional regulation of the PTTH receptor in prothoracic glands of the silkworm, Bombyx mori.

    PubMed

    Young, Shun-Chieh; Yeh, Wei-Lan; Gu, Shi-Hong

    2012-01-01

    The present study investigated transcriptional regulation of the prothoracicotropic hormone (PTTH) receptor (Torso) gene in prothoracic glands (PGs) of the silkworm, Bombyx mori. The results showed that PTTH treatment in vitro time-dependently affected Torso mRNA levels, with an inhibitory effect being detected after 1- and 2-h periods of incubation. When methoprene, a juvenile hormone analogue (JHA), was applied to newly ecdysed last instar larvae, a decline in Torso mRNA levels during the early last larval instar was delayed compared to those treated with acetone. Injection of 20-hydroxyecdysone appeared to have a stimulatory effect on Torso mRNA levels. Torso mRNA levels were also shown to be nutrition-sensitive. From these results, it was suggested that Torso mRNA levels of the PGs appear to be hormonally regulated and nutrition-sensitive, and the endogenous precisely coordinated regulation of Torso mRNA levels may play a role in regulating ecdysteroidogenesis by PGs during development. Copyright © 2011. Published by Elsevier Ltd.

  7. Brain factor-7 extracted from Bombyx mori enhances cognition and attention in normal children.

    PubMed

    Kim, Kunwoo; Park, Subin; Yoo, Hanik K; Lee, Jun-Young; Jung, Hee-Yeon; Kim, Do-Hee; Lee, Hyun-Jung; Kim, Jin-Young; Youn, Young-Chul; Marshall, Maurice R; Kim, Sung-Su; Jeong, Yoonhwa

    2009-06-01

    It has been reported that brain factor-7 (BF-7) extracted from Bombyx mori improves cognitive functions in normal juveniles and adults as well as cognitively impaired patients. Clinical studies with normal children evaluated the role of BF-7 on brain function in these patients. The objective of this study was to improve cognitive functions of normal schoolchildren with BF-7. Forty-six normal healthy children were divided into two treatment groups: BF-7 (9.9 +/- 1.18 years old; 9 boys, 14 girls) and placebo (9.8 +/- 1.03 years old; 10 boys, 13 girls). The Color Trails Making Test was used to measure the efficacy of BF-7 on cognition and attention. Results showed that BF-7 reduced the response time by an average of 23% for the Color Trails Making Test. Moreover, BF-7 improved the accuracy of the task around twofold. The results reveal that BF-7 improves brain function for attention and cognitive flexibility in children.

  8. Proteomic and immunological identification of two new allergens from silkworm (Bombyx mori L.) pupae.

    PubMed

    Zhao, Xiangjie; Li, Lin; Kuang, Zheshi; Luo, Guoqing; Li, Bing

    2015-01-01

    This study explored food allergy caused by eating silkworm (Bombyx mori L.) pupae, a traditionally accepted food and animal feed in East and Southeast Asia, and identified two new allergens by proteomic and immunological methods. Proteins isolated from silkworm pupae were separated by two-dimensional gel electrophoresis (2-DE); pooled sera from patients allergic to silkworm pupa proteins were used to detect immunoglobulin E (IgE)-binding proteins by western blotting, and allergens specific for silkworm pupa consumption-caused allergy were visualised with the ECL reagents. The selected allergen proteins were further identified by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. Finally, chitinase and paramyosin were identified as silkworm pupa proteins showing strong immunoglobulin (IgE)-binding reaction. Analysis of the sequence homology of the two proteins using the AllergenOnline database indicated that chitinase and paramyosin shared 24.8% and 62.8% sequence homology with known allergens Der f 18 (Dermatophagoides farinae) and Der p 11 (Dermatophagoides pteronyssinus), respectively. Our results shed light on the understanding and treatment of silkworm pupa allergy.

  9. Changes in lipid droplet localization during embryogenesis of the silkworm, Bombyx mori.

    PubMed

    Yamahama, Yumi; Seno, Keiji; Hariyama, Takahiko

    2008-06-01

    Lipid droplets are considered one of the most important energy sources in lepidopteran eggs during late embryogenesis, but the process of their incorporation into the embryo is as yet unknown. The present study focused on the process of transition of lipid droplets from the extraembryonic yolk to the embryo of the silkworm Bombyx mori, using morphological and biochemical approaches. The morphological study revealed that the incorporation of lipid droplets from the extraembryonic yolk into the embryo occurs at three points and in three different ways during the development of the embryo. Some lipid droplets were translocated directly from the extraembryonic yolk to the embryo before the blastokinesis stage. However, the majority of lipid droplets together with the other components of the extraembryonic yolk were incorporated in the embryo via both morphogenetic inclusion during dorsal closure and ingestion of the extraembyonic yolk by the developing caterpillar prior to hatching. Similar results were obtained from the biochemical study. Thus, we propose that there are three steps in the incorporation of lipid droplets from the extraembryonic yolk into the embryo. In addition, morphological and biochemical data concerning the total amount of lipid droplets in the egg suggested that lipid droplets were mainly consumed during late embryogenesis, seeming to synchronize with tracheal development.

  10. Bm65 is essential for the propagation of Bombyx mori nucleopolyhedrovirus.

    PubMed

    Tang, Qi; Li, Guohui; Yao, Qin; Chen, Liang; Feng, Fan; Yuan, Yi; Chen, Keping

    2013-01-01

    Orf65 (Bm65) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene that encodes an unknown 104-amino acid protein. In the present study, we have shown the role of Bm65 in the baculovirus life cycle. 5'-RACE analysis showed that the transcription start site of Bm65 was 14 nucleotides upstream of the start codon ATG. The transcription profile of Bm65 was detected from 6 to 72 h postinfection (p. i.) by RT-PCR. A Bm65-knockout bacmid was constructed by homologous recombination to characterize the role of Bm65 in viral life cycle. Fluorescence microscopy showed that Bm65-knockout virus was unable to generate infectious budded virus in BmN cells. Furthermore, quantitative real-time PCR analysis demonstrated that Bm65 deletion did not affect the viral DNA replication. To conclude, Bm65 is essential for the propagation of BmNPV, but is unnecessary for the replication of viral DNA.

  11. Genetic characterization of Bombyx mori (Lepidoptera: Bombycidae) breeding and hybrid lines with different geographic origins.

    PubMed

    Furdui, Emilia M; Mărghitaş, Liviu A; Dezmirean, Daniel S; Paşca, Ioan; Pop, Iulia F; Erler, Silvio; Schlüns, Ellen A

    2014-01-01

    The domesticated silkworm Bombyx mori L. comprises a large number of geographical breeds and hybrid lines. Knowing the genetic structure of those may provide information to improve the conservation of commercial lines by estimating inbreeding over generations and the consequences of excessive use of those lineages. Here, we analyzed the genetic diversity of seven breeds and eight hybrid lines from Eastern Europe and Asia using highly polymorphic microsatellites markers to determine its genetical impact on their use in global breeding programs. No consistent pattern of deviation from Hardy-Weinberg equilibrium was found for most breed and hybrids; and the absence of a linkage disequilibrium also suggests that the strains are in equilibrium. A principal coordinate analysis revealed a clear separation of two silkworm breeds from the rest: one (IBV) originated from India and the other one (RG90) from Romania/Japan. The tendency of the other breeds from different geographic origins to cluster together in a general mix might be due to similar selection pressures (climate and anthropogenic factors) in different geographic locations. Phylogenetic analyses grouped the different silkworm breeds but not the hybrids according to their geographic origin and confirmed the pattern found in the principal coordinate analysis. © The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America.

  12. Evidence for Nucleic Acid Binding Ability and Nucleosome Association of Bombyx mori Nucleopolyhedrovirus BRO Proteins

    PubMed Central

    Zemskov, Evgueni A.; Kang, WonKyung; Maeda, Susumu

    2000-01-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) genome contains five related members of the bro gene family, all of which are actively expressed in infected BmN cells. Although their functions are unknown, their amino acid sequences contain a motif found in all known viral and prokaryotic single-stranded DNA binding proteins. To determine if they bind to nucleic acids, we fractionated the nuclei of BmNPV-infected BmN cells using a histone extraction protocol. We detected BRO-A, BRO-C, and BRO-D in the histone H1 fraction using anti-BRO antibodies. Micrococcal nuclease treatment released these BRO proteins from the chromatin fraction, suggesting their involvement in nucleosome structures. Chromatographic fractionation showed that BRO-A and/or BRO-C interacted with core histones. Expression of partial sequences of BRO-A proved that the N-terminal 80 amino acid residues were required for DNA binding activity. We also demonstrated that BmNPV BRO proteins underwent phosphorylation and ubiquitination followed by proteasome degradation, which may explain their distribution in the cytoplasm as well as the nucleus. We propose that BRO-A and BRO-C may function as DNA binding proteins that influence host DNA replication and/or transcription. PMID:10888617

  13. Combinatory annotation of cell membrane receptors and signalling pathways of Bombyx mori prothoracic glands

    PubMed Central

    Moulos, Panagiotis; Samiotaki, Martina; Panayotou, George; Dedos, Skarlatos G.

    2016-01-01

    The cells of prothoracic glands (PG) are the main site of synthesis and secretion of ecdysteroids, the biochemical products of cholesterol conversion to steroids that shape the morphogenic development of insects. Despite the availability of genome sequences from several insect species and the extensive knowledge of certain signalling pathways that underpin ecdysteroidogenesis, the spectrum of signalling molecules and ecdysteroidogenic cascades is still not fully comprehensive. To fill this gap and obtain the complete list of cell membrane receptors expressed in PG cells, we used combinatory bioinformatic, proteomic and transcriptomic analysis and quantitative PCR to annotate and determine the expression profiles of genes identified as putative cell membrane receptors of the model insect species, Bombyx mori, and subsequently enrich the repertoire of signalling pathways that are present in its PG cells. The genome annotation dataset we report here highlights modules and pathways that may be directly involved in ecdysteroidogenesis and aims to disseminate data and assist other researchers in the discovery of the role of such receptors and their ligands. PMID:27576083

  14. Effect of Bombyx mori chitinase against Japanese pine sawyer (Monochamus alternatus) adults as a biopesticide.

    PubMed

    Kabir, Khondkar Ehteshamul; Sugimoto, Hiroyuki; Tado, Hiroyuki; Endo, Katsuhiko; Yamanaka, Akira; Tanaka, Shuhei; Koga, Daizo

    2006-01-01

    Bombyx mori chitinase (Bm-CHI), with a molecular mass of 75 kDa, was investigated on the possibility that it can serve as a biocontrol agent against the adult Japanese pine sawyer (JPS), Monochamus alternatus (Coleoptera: Cerambycidae). Oral ingestion of purified chitinase at concentrations of 3 microM (11.25 microg/50 microl) and 0.3 micoM (1.125 microg/50 microl) caused high mortality in JPS, a significant decrease in bark consumption, and, only in high concentration, a slight reduction of body weight. Fluorescence assays indicated that peritrophic membrane (PM) chitin is degraded by the action of orally ingested Bm-CHI at 3 microM concentration only. Scanning electron micrographs clearly indicated that the beetles that ingested Bm-CHI of the same high concentration had their PM perforated and disrupted, but ultrastructural studies showed that the ingested chitinase did not affect the midgut epithelium. These findings open up the possibility of using insect chitinase as a biopesticidal enzyme. It should have agronomic potential for insect control.

  15. Anticancer activity of Bombyx batryticatus ethanol extract against the human tumor cell line HeLa.

    PubMed

    Wu, W P; Cao, J; Wu, J Y; Chen, H; Wang, D

    2015-01-15

    Anticancer activity of Bombyx batryticatus ethanol extract (BBE) against HeLa cells was studied using cell viability, DNA fragmentation, real-time polymerase chain reaction, and Western blot analyses. The BBE inhibited the growth and induced apoptosis of HeLa cells. The MTT assay indicated that the BBE induced cytotoxicity in HeLa cells in a time- and concentration-dependent manner. When HeLa cells were treated for 48 h, the 50% inhibitory concentration (IC₅₀) value for the BBE was 1.564 mg/mL. The microscopy results showed that HeLa cells were severely distorted and showed slow growth; some cells became round in shape when treated with 5 mg/mL BBE for 24 h. The DNA ladder results revealed excessive DNA fragmentation in HeLa cells treated with 7 mg/mL BBE for 36 h. The proapoptotic activity of the BBE was attributed to its ability to modulate the expression of Bcl-2 and Bax genes. The mRNA and protein expression levels of Bax were remarkably higher whereas those of Bcl-2 were lower than those in the control cells; this led to an increased Bax/Bcl-2 ratio in cells treated with the BBE for 36 h. The results suggest that the BBE might play an important role in tumor growth suppression by inducing apoptosis in human cervical cancer cells via the regulation of the Bcl-2- and Bax-mediated apoptotic pathways.

  16. Electroporation, an alternative to biolistics for transfection of Bombyx mori embryos and larval tissues

    PubMed Central

    Thomas, Jean-luc

    2003-01-01

    There are few powerful techniques available to transfect insect tissues. We previously used biolistics to transfect Bombyx mori embryos, and larval and pupal tissues (Thomas J-L et al. 2001. Journal of Insect Science 1/9, Kravariti L et al. 2001. Insect Biochemistry and Molecular Biology 31: 473–479). As the main limitation was the irregularity in results we explored electroporation as an alternative technique by adapting techniques used for chicken embryos to B. mori embryos. By injecting the DNA solution into the hemocoel of late embryos that were finishing organogenesis, we expressed marker genes in numerous tissues following electroporation. With some adaptation of the method this was also achieved for early embryos lacking a hemocoel. Some larval tissues were also transfected. During these technical studies we found that optimizing parameters such as electrical voltage, number of pulses and their frequency, and conductivity of the buffer was important. These results confirmed that electroporation is a reliable technique for transfecting B. mori tissues. Abbreviation: GFP Green Fluorescent Protein CCD Charged Coupled Device PMID:15841233

  17. Enhancing Effect of Glycerol on the Tensile Properties of Bombyx mori Cocoon Sericin Films

    PubMed Central

    Zhang, Haiping; Deng, Lianxia; Yang, Mingying; Min, Sijia; Yang, Lei; Zhu, Liangjun

    2011-01-01

    An environmental physical method described herein was developed to improve the tensile properties of Bombyx mori cocoon sericin films, by using the plasticizer of glycerol, which has a nontoxic effect compared with other chemical crosslinkers. The changes in the tensile characteristics and the structure of glycerolated (0–40 wt% of glycerol) sericin films were investigated. Sericin films, both in dry and wet states, showed enhanced tensile properties, which might be regulated by the addition of different concentrations of glycerol. The introduction of glycerol results in the higher amorphous structure in sericin films as evidenced by analysis of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra, thermogravimetry (TGA) and differential scanning calorimetry (DSC) curves. Scanning Electron Microscopy (SEM) observation revealed that glycerol was homogeneously blended with sericin molecules when its content was 10 wt%, while a small amount of redundant glycerol emerged on the surface of sericin films when its content was increased to 20 wt% or higher. Our results suggest that the introduction of glycerol is a novel nontoxic strategy which can improve the mechanical features of sericin-based materials and subsequently promote the feasibility of its application in tissue engineering. PMID:21686177

  18. Microarray analysis of New Green Cocoon associated genes in silkworm, Bombyx mori.

    PubMed

    Lu, Ya-Ru; He, Song-Zhen; Tong, Xiao-Ling; Han, Min-Jin; Li, Chun-Lin; Li, Zhi-Quan; Dai, Fang-Yin

    2016-06-01

    Green cocoons in silkworm, Bombyx mori, are caused by flavonoids accumulation in the silk proteins, fibroin and sericin. Despite the economic value of natural green cocoon and medical value of flavonoids, there is limited understanding of the molecular mechanism regulating flavonoids uptake in silkworm, which is tightly associated with the trait of green cocoon. The purpose of this study is to perform a comprehensive analysis to understand the molecular mechanisms of flavonoids uptake in silkworm based on microarray analyses. The study subject was the New Green Cocoon from the silkworm strains, G200 and N100, a new spontaneous dominant green cocoon trait identified in the 2000s. The genes regulating this trait are independent of other green cocoon genes previously reported. Genome-wide gene expression was compared between the New Green Cocoon producing silkworm strains, G200 and N100, and the control sample, which is the white cocoon producing strain 872B. Among these strains, N100 and 872B are near-isogenic lines. The results showed that 130 genes have consistently changing expression patterns in the green cocoon strains when compared with the white cocoon strain. Among these, we focused on the genes related to flavonoids metabolism and absorption, such as sugar transporter genes and UDP-glucosyltransferase genes. Based on our findings, we propose the potential mechanisms for flavonoids absorption and metabolism in silkworm. Our results imply that silkworm might be used as an underlying model for flavonoids in pharmaceutical research.

  19. Appearance of differentiated cells derived from polar body nuclei in the silkworm, Bombyx mori.

    PubMed

    Sakai, Hiroki; Yokoyama, Takeshi; Abe, Hiroaki; Fujii, Tsuguru; Suzuki, Masataka G

    2013-01-01

    In Bombyx mori, polar body nuclei are observed until 9 h after egg lying, however, the fate of polar body nuclei remains unclear. To examine the fate of polar body nuclei, we employed a mutation of serosal cell pigmentation, pink-eyed white egg (pe). The heterozygous pe/+ (pe) females produced black serosal cells in white eggs, while pe/pe females did not produce black serosal cells in white eggs. These results suggest that the appearance of black serosal cells in white eggs depends on the genotype (pe/+ (pe) ) of the mother. Because the polar body nuclei had + (pe) genes in the white eggs laid by a pe/+ (pe) female, polar body nuclei participate in development and differentiate into functional cell (serosal cells). Analyses of serosal cells pigmentation indicated that ~30% of the eggs contained polar-body-nucleus-derived cells. These results demonstrate that polar-body-nucleus-derived cells appeared at a high frequency under natural conditions. Approximately 80% of polar-body-nucleus-derived cells appeared near the anterior pole and the dorsal side, which is opposite to where embryogenesis occurs. The number of cells derived from the polar body nuclei was very low. Approximately 26% of these eggs contained only one black serosal cell. PCR-based analysis revealed that the polar-body-nucleus-derived cells disappeared in late embryonic stages (stage 25). Overall, polar-body-nuclei-derived cells were unlikely to contribute to embryos.

  20. Analysis of proteome dynamics inside the silk gland lumen of Bombyx mori.

    PubMed

    Dong, Zhaoming; Zhao, Ping; Zhang, Yan; Song, Qianru; Zhang, Xiaolu; Guo, Pengchao; Wang, Dandan; Xia, Qingyou

    2016-04-22

    The silk gland is the only organ where silk proteins are synthesized and secreted in the silkworm, Bombyx mori. Silk proteins are stored in the lumen of the silk gland for around eight days during the fifth instar. Determining their dynamic changes is helpful for clarifying the secretion mechanism of silk proteins. Here, we identified the proteome in the silk gland lumen using liquid chromatography-tandem mass spectrometry, and demonstrated its changes during two key stages. From day 5 of the fifth instar to day 1 of wandering, the abundances of fibroins, sericins, seroins, and proteins of unknown functions increased significantly in different compartments of the silk gland lumen. As a result, these accumulated proteins constituted the major cocoon components. In contrast, the abundances of enzymes and extracellular matrix proteins decreased in the silk gland lumen, suggesting that they were not the structural constituents of silk. Twenty-five enzymes may be involved in the regulation of hormone metabolism for proper silk gland function. In addition, the metabolism of other non-proteinous components such as chitin and pigment were also discussed in this study.

  1. Metabolic allometry during development and metamorphosis of the silkworm Bombyx mori: analyses, patterns, and mechanisms.

    PubMed

    Blossman-Myer, Bonnie L; Burggren, Warren W

    2010-01-01

    Intraspecific allometric (scaling) relationships for metabolism, which have received little examination compared to interspecific relationships, reflect a complex interplay of organogenesis, growth, and shifting physiologies. In this study of the silkworm Bombyx mori, we hypothesized that allometric relationships for metabolism both across all developmental stages and within each stage would not reflect conventional scaling coefficients (e.g., b not equal to 0.75). Histology, gross morphology, body surface and cross-sectional area, total lipid content, and cytochrome c oxidase activity levels (as evidence of the total metabolic potential of mitochondria) were determined across development. Also measured were oxygen consumption, carbon dioxide production, and the respiratory exchange ratio. The overall slope, b, in the allometric equation relating to body mass across all developmental stages was 0.82, not greatly different from the value of 0.75 typical of interspecific data. However, within larval instars II-V and in prepupae, b varied between 0.99 and 1.49, far higher than hypothesized. Thus, in B. mori, an analytical approach that lumps all developmental stages hides interinstar variability. Morphological and biochemical data suggest that observed scaling patterns in B. mori are likely correlated with changes in overall mitochondrial density rather than with specific changes in body proportion of tissues with higher intrinsic metabolic intensity.

  2. Appearance of differentiated cells derived from polar body nuclei in the silkworm, Bombyx mori

    PubMed Central

    Sakai, Hiroki; Yokoyama, Takeshi; Abe, Hiroaki; Fujii, Tsuguru; Suzuki, Masataka G.

    2013-01-01

    In Bombyx mori, polar body nuclei are observed until 9 h after egg lying, however, the fate of polar body nuclei remains unclear. To examine the fate of polar body nuclei, we employed a mutation of serosal cell pigmentation, pink-eyed white egg (pe). The heterozygous pe/+pe females produced black serosal cells in white eggs, while pe/pe females did not produce black serosal cells in white eggs. These results suggest that the appearance of black serosal cells in white eggs depends on the genotype (pe/+pe) of the mother. Because the polar body nuclei had +pe genes in the white eggs laid by a pe/+pe female, polar body nuclei participate in development and differentiate into functional cell (serosal cells). Analyses of serosal cells pigmentation indicated that ~30% of the eggs contained polar-body-nucleus-derived cells. These results demonstrate that polar-body-nucleus-derived cells appeared at a high frequency under natural conditions. Approximately 80% of polar-body-nucleus-derived cells appeared near the anterior pole and the dorsal side, which is opposite to where embryogenesis occurs. The number of cells derived from the polar body nuclei was very low. Approximately 26% of these eggs contained only one black serosal cell. PCR-based analysis revealed that the polar-body-nucleus-derived cells disappeared in late embryonic stages (stage 25). Overall, polar-body-nuclei-derived cells were unlikely to contribute to embryos. PMID:24027530

  3. Midgut immune responses induced by bacterial infection in the silkworm, Bombyx mori *

    PubMed Central

    ZHANG, Lei; WANG, Yan-wen; LU, Zhi-qiang

    2015-01-01

    Insect gut epithelial cells produce reactive oxygen species (ROS) and antimicrobial peptides (AMPs) to protect hosts from pathogenic microorganisms. In this study, we evaluate the pathogenicity of Pseudomonas aeruginosa and Bacillus bombysepticus in the silkworm, Bombyx mori. Survival curves show that B. bombysepticus is deadly when larval silkworms are infected orally. Bacterial infection caused intestinal hydrogen peroxide (H2O2) and nitric oxide (NO) levels to increase significantly by 8 and 16 h post-infection (hpi), respectively. Real-time quantitative polymerase chain reaction (qPCR) analysis shows that the transcription levels of dual oxidase (Duox) and catalase (CAT) are highly up-regulated by P. aeruginosa infection at 8 hpi. P. aeruginosa infection induced nitric oxide synthase 2 (NOS2) expression at 16 hpi, which contributes to the generation of NO. mRNA levels of AMP genes, specifically Glovorin 2 and Glovorin 3, which obviously increase during the early infection stage. These results indicate that invading bacteria elevate intestinal ROS and NO levels and induce AMP gene transcription, which contributes to intestinal immune defense. PMID:26465135

  4. Identification and molecular characterization of a chitin deacetylase from Bombyx mori peritrophic membrane.

    PubMed

    Zhong, Xiao-Wu; Wang, Xiao-Huan; Tan, Xiang; Xia, Qing-You; Xiang, Zhong-Huai; Zhao, Ping

    2014-01-27

    The insect midgut epithelium is generally lined with a unique chitin and protein structure, the peritrophic membrane (PM), which facilitates food digestion and protects the gut epithelium. PM proteins are important determinants for PM structure and formation. In this study, the silkworm Bombyx mori midgut PM protein BmCDA7 was identified by proteomic tools. The full-length BmCDA7 cDNA is 1357 bp; the deduced protein is composed of 379 amino acid residues and includes a 16 amino acid residue signal peptide, a putative polysaccharide deacetylase-like domain and 15 cysteine residues present in three clusters. The heterologously expressed proteins of the BmCDA7 gene in yeast displayed chitin deacetylase activity. Expression of B. mori BmCDA7 was detected in the midgut at both the transcriptional and translational levels. The BmCDA7 gene was expressed by the newly hatched silkworm larvae until day seven of the fifth instar and was expressed at a high level in the newly exuviated larvae of different instars. The functions and regulatory mechanism of BmCDA7, however, need further investigation.

  5. Developmental Changes for the Hemolymph Metabolome of Silkworm (Bombyx moriL.)

    PubMed Central

    Zhou, Lihong; Li, Huihui; Hao, Fuhua; Li, Ning; Liu, Xin; Wang, Guoliang; Wang, Yulan; Tang, Huiru

    2015-01-01

    Silkworm (Bombyx mori) is a lepidopteran-holometabolic model organism. To understand its developmental biochemistry, we characterized the larval hemolymph metabonome from the third instar to prepupa stage using 1H NMR spectroscopy whilst hemolymph fatty acid composition using GC-FID/MS. We unambiguously assigned more than 60 metabolites, among which tyrosine-o-β-glucuronide, mesaconate, homocarnosine, and picolinate were reported for the first time from the silkworm hemolymph. Phosphorylcholine was the most abundant metabolite in all developmental stages with exception for the periods before the third and fourth molting. We also found obvious developmental dependence for the hemolymph metabonome involving multiple pathways including protein biosyntheses, glycolysis, TCA cycle, the metabolisms of choline amino acids, fatty acids, purines, and pyrimidines. Most hemolymph amino acids had two elevations during the feeding period of the fourth instar and prepupa stage. Trehalose was the major blood sugar before day 8 of the fifth instar, whereas glucose became the major blood sugar after spinning. C16:0, C18:0 and its unsaturated forms were dominant fatty acids in hemolymph. The developmental changes of hemolymph metabonome were associated with dietary nutrient intakes, biosyntheses of cell membrane, pigments, proteins, and energy metabolism. These findings offered essential biochemistry information in terms of the dynamic metabolic changes during silkworm development. PMID:25825269

  6. Comparative Proteome Analysis of Multi-Layer Cocoon of the Silkworm, Bombyx mori

    PubMed Central

    Dong, Zhaoming; Wang, Dandan; Guo, Pengchao; Guo, Xiaomeng; Song, Qianru; Zhang, Weiwei; Xia, Qingyou

    2015-01-01

    Bombyx mori cocoon has a multi-layer structure that provides optimal protection for silkworm pupa. Research on the mechanical properties of the multi-layer structure revealed structure-property relationships of the cocoon. Here, we investigated the protein components of the B. mori cocoon in terms of its multi-layer structure. Liquid chromatography-tandem mass spectrometry identified 286 proteins from the multiple cocoon layers. In addition to fibroins and sericins, we identified abundant protease inhibitors, seroins and proteins of unknown function. By comparing protein abundance across layers, we found that the outermost layer contained more sericin1 and protease inhibitors and the innermost layer had more seroin1. As many as 36 protease inhibitors were identified in cocoons, showing efficient inhibitory activities against a fungal protease. Thus, we propose that more abundant protease inhibitors in the outer cocoon layers may provide better protection for the cocoon. This study increases our understanding of the multi-layer mechanism of cocoons, and helps clarify the biological characteristics of cocoons. The data have been deposited to the ProteomeXchange with identifier PXD001469. PMID:25860555

  7. Cloning, expression and characterization of Bombyx mori α1,6-fucosyltransferase.

    PubMed

    Ihara, Hideyuki; Okada, Takahiro; Ikeda, Yoshitaka

    2014-07-25

    Although core α1,6-fucosylation is commonly observed in N-glycans of both vertebrates and invertebrates, the responsible enzyme, α1,6-fucosyltransferase, has been much less characterized in invertebrates compared to vertebrates. To investigate the functions of α1,6-fucosyltransferase in insects, we cloned the cDNA for the α1,6-fucosyltransferase from Bombyx mori (Bmα1,6FucT) and characterized the recombinant enzyme prepared using insect cell lines. The coding region of Bmα1,6FucT consists of 1737bp that code for 578 amino acids of the deduced amino acid sequence, showing significant similarity to other α1,6-fucosyltransferases. Enzyme activity assays demonstrated that Bmα1,6FucT is enzymatically active in spite of being less active compared to the human enzyme. The findings also indicate that Bmα1,6FucT, unlike human enzyme, is N-glycosylated and forms a disulfide-bonded homodimer. These findings contribute to a better understanding of roles of α1,6-fucosylation in invertebrates and also to the development of the more efficient engineering of N-glycosylation of recombinant glycoproteins in insect cells.

  8. Activation of Bombyx neuropeptide G protein-coupled receptor A4 via a Gαi-dependent signaling pathway by direct interaction with neuropeptide F from silkworm, Bombyx mori.

    PubMed

    Deng, Xiaoyan; Yang, Huipeng; He, Xiaobai; Liao, Yuan; Zheng, Congxia; Zhou, Qi; Zhu, Chenggang; Zhang, Guozheng; Gao, Jimin; Zhou, Naiming

    2014-02-01

    Members of the mammalian neuropeptide Y (NPY) family serve as neurotransmitters and contribute to a diversity of physiological functions. Although neuropeptide F (NPF), the NPY-like orthologs from insects, have been identified, the NPF receptors and their signaling and physiological functions remain largely unknown. In this study, we established the stable and transient functional expression of a Bombyx orphan G protein-coupled receptor, BNGR-A4, in both mammalian HEK293 and insect SF21 cells. We identified Bombyx mori NPFs as specific endogenous ligands for the Bombyx Neuropeptide GPCR A4 (BNGR-A4) and, accordingly, named the receptor BomNPFR. Our results demonstrated that BomNPFR was activated by synthetic BomNPF1a and BomNPF1b at a high efficacy and by BomNPF2 at a low efficacy. This activation led to a decrease of forskolin or adipokinetic hormone peptide-stimulated adenylyl cyclase activity, an increase of intracellular Ca(2+), the activation of ERK1/2 signaling and receptor internalization. Moreover, a Rhodamine-labeled BomNPF1a peptide was found to bind specifically to BomNPFR. The results derived from quantitative RT-PCR analysis and dsRNA-mediated knockdown experiments demonstrated the possible role of BomNPFR in the regulation of food intake and growth. Our results provide the first in-depth information on BomNPFR-mediated signaling for the further elucidation of the BomNPF/BomNPFR system in the regulation of fundamental physiological processes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Effects of insect viruses and pesticides on glutathione S-transferase activity and gene expression in Bombyx mori.

    PubMed

    Gui, Zhongzheng; Hou, Chengxiang; Liu, Ting; Qin, Guangxin; Li, Muwang; Jin, Byungrae

    2009-08-01

    Glutathione S-transferase (GST) is a gene family generally associated with detoxification and plays an important role in detoxifying exogenous compounds. The silkworm Bombyx mori is an important economic animal for silk production. However, it is liable to infection by a number of viruses and chemical agents that can contaminate its food and growing environment. Here we conducted a comparative study of strain- and tissue-specific expressions of GST in the silkworm under infections by Bombyx mori nuclear polyhedrosis virus (BmNPV) and Bombyx mori densonucleosis virus (BmDNV) and under the stress of pesticides (insecticide and herbicide). BmDNV induced an increase of GST at 24 h and a decrease at 48 and 72 h in the midgut of the DNV-susceptible strain and a decrease at 24 h and increase at 48 and 72 h in the midgut of the DNV-tolerant strain. BmDNV induced a significant increase of GST from 24 to 72 h in the fat body of both DNV-susceptible and DNV-tolerant strains. In contrast, BmNPV induced a significant decrease of GST in both the fat body and midgut of the NPV-susceptible strain and induced increase of GST from 24 to 48 h in the midgut and at 72 h in the fat body of the NPV-tolerant strain. All of these results suggest that BmGST activity varies with the strain, tissue, feeding behavior, and developmental stage of the silkworm. On treating silkworm larvae with pesticides (insecticide and herbicide), expression of the BmGSTS2 gene increased noticeably in the midgut and reached a peak at 6 to 12 h. The mRNA level of BmGSTS2 in the midgut increased during administration of the chemicals, suggesting that the induction of BmGSTS2 is part of the defense mechanism against exogenous chemicals.

  10. Screening of candidate proteins interacting with IE-2 of Bombyx mori nucleopolyhedrovirus.

    PubMed

    Wu, Yejun; Wu, Yu; Wu, Yan; Tang, Hui; Wu, Huiling; Zhang, Guozheng; Wang, Wenbing

    2013-10-01

    IE-2 of Bombyx mori nucleopolyhedrovirus (BmNPV) has been shown to play important roles in baculovirus infection, which are involved in gene expression and viral replication. However, the mechanism remains unknown. In this paper, by TargetP software, four genes, i.e.-2, odv-e26, odv-e56 and BmNPV-gp101 (Ac-orf116) of BmNPV and Autographa californica multiple NPV (AcMNPV) were predicted to be located in mitochondria. By BLAST tool using BmNPV IE-2 protein sequence, 14 NPVs were found to have IE-2 homologues in GenBank, and most of them were predicted to be located in mitochondria, except for that of Antheraea pernyi NPV (AnpeNPV) and Anticarsia gemmatalis NPV (AngeNPV). To observe the subcellular localization of BmNPV IE-2, a recombinant virus overexpressed the IE-2 and eGFP fusion protein was constructed. In infected BmN cells, the fluorescence specifically enriched in the cellular mitochondria. This evidence was accordant with the prediction. Further, Pull-down assay was used to select protein candidates interacting with IE-2 in B. mori cells infected with BmNPV. Of several isolated protein components, sixteen candidates were identified by MALDI-TOF mass-spectrometry, eight baculoviral proteins (ALK-EXO, F protein, IAP-1, LEF-3, LEF-9, ODV-NC42, TLP, and VP39), and eight proteins from B. mori (Actin, ADP/ATP translocase, ATP synthase subunit beta, Beta-tubulin, DNA topoisomerase 2, Histone H4, Soluble guanylyl cyclae alpha-1 subunit, Transketolase). From the functional point of view, most of these proteins were generally divided into two groups, mitochondrial interaction proteins and viral DNA replication proteins. These results implied that the IE-2 had multiple functions involved in regulating viral gene expression, viral replication and also as a component of mitochondrial factors to regulate the cellular energy supply and apoptosis.

  11. Expression pattern of immunoglobulin superfamily members in the silkworm, Bombyx mori.

    PubMed

    He, Lei; Cao, Guangli; Huang, Moli; Xue, Renyu; Hu, Xiaolong; Gong, Chengliang

    2014-09-15

    Immunoglobulin superfamily (IgSF) proteins are involved in cell adhesion, cell communication and immune functions. In this study, 152 IgSF genes containing at least one immunoglobulin (Ig) domain were predicted in the Bombyx mori silkworm genome. Of these, 145 were distributed on 25 chromosomes with no genes on chromosomes 16, 18 and 26. Multiple sequence alignments and phylogenetic evolution analysis indicated that IgSFs evolved rapidly. Gene ontology (GO) annotation indicated that IgSF members functioned as cellular components and in molecular functions and biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that IgSF proteins were involved in signal transduction, signaling molecules and interaction, and cell communication. Microarray-based expression data showed tissue expression for 136 genes in anterior silkgland, middle silkgland, posterior silkgland, testis, ovary, fat body, midgut, integument, hemocyte, malpighian tubule and head. Expression pattern of IgSF genes in the silkworm ovary and midgut was analyzed by RNA-Seq. Expression of 105 genes was detected in the ovary in strain Dazao. Expression in the midgut was detected for 74 genes in strain Lan5 and 75 genes in strain Ou17. Expression of 34 IgSF genes in the midgut relative to the actin A3 gene was significantly different between strains Lan5 and Ou17. Furthermore, 1 IgSF gene was upregulated and 1 IgSF gene was downregulated in strain Lan5, and 4 IgSF genes were upregulated and 2 IgSF genes were downregulated in strain Ou17 after silkworms were challenged with B. mori cypovirus (BmCPV), indicating potential involvement in the response to BmCPV-infection. These results provide an overview of IgSF family members in silkworms, and lay the foundation for further functional studies.

  12. The genome of a lepidopteran model insect, the silkworm Bombyx mori.

    PubMed

    2008-12-01

    Bombyx mori, the domesticated silkworm, is a major insect model for research, and the first lepidopteran for which draft genome sequences became available in 2004. Two independent data sets from whole-genome shotgun sequencing were merged and assembled together with newly obtained fosmid- and BAC-end sequences. The remarkably improved new assembly is presented here. The 8.5-fold sequence coverage of an estimated 432 Mb genome was assembled into scaffolds with an N50 size of approximately 3.7 Mb; the largest scaffold was 14.5 million base pairs. With help of a high-density SNP linkage map, we anchored 87% of the scaffold sequences to all 28 chromosomes. A particular feature was the high repetitive sequence content estimated to be 43.6% and that consisted mainly of transposable elements. We predicted 14,623 gene models based on a GLEAN-based algorithm, a more accurate prediction than the previous gene models for this species. Over three thousand silkworm genes have no homologs in other insect or vertebrate genomes. Some insights into gene evolution and into characteristic biological processes are presented here and in other papers in this issue. The massive silk production correlates with the existence of specific tRNA clusters, and of several sericin genes assembled in a cluster. The silkworm's adaptation to feeding on mulberry leaves, which contain toxic alkaloids, is likely linked to the presence of new-type sucrase genes, apparently acquired from bacteria. The silkworm genome also revealed the cascade of genes involved in the juvenile hormone biosynthesis pathway, and a large number of cuticular protein genes.

  13. Establishment of Tools for Neurogenetic Analysis of Sexual Behavior in the Silkmoth, Bombyx mori

    PubMed Central

    Kiya, Taketoshi; Morishita, Koudai; Uchino, Keiro; Iwami, Masafumi; Sezutsu, Hideki

    2014-01-01

    Background Silkmoth, Bombyx mori, is an ideal model insect for investigating the neural mechanisms underlying sex pheromone-induced innate behavior. Although transgenic techniques and the GAL4/UAS system are well established in the silkmoth, genetic tools useful for investigating brain function at the neural circuit level have been lacking. Results In the present study, we established silkmoth strains in which we could visualize neural projections (UAS-mCD8GFP) and cell nucleus positions (UAS-GFP.nls), and manipulate neural excitability by thermal stimulation (UAS-dTrpA1). In these strains, neural projections and nucleus position were reliably labeled with green fluorescent protein in a GAL4-dependent manner. Further, the behavior of silkworm larvae and adults could be controlled by GAL4-dependent misexpression of dTrpA1. Ubiquitous dTrpA1 misexpression led both silkmoth larvae and adults to exhibit seizure-like phenotypes in a heat stimulation-dependent manner. Furthermore, dTrpA1 misexpression in the sex pheromone receptor neurons of male silkmoths allowed us to control male sexual behavior by changing the temperature. Thermally stimulated male silkmoths exhibited full sexual behavior, including wing-flapping, orientation, and attempted copulation, and precisely approached a thermal source in a manner similar to male silkmoths stimulated with the sex pheromone. Conclusion These findings indicate that a thermogenetic approach using dTrpA1 is feasible in Lepidopteran insects and thermogenetic analysis of innate behavior is applicable in the silkmoth. These tools are essential for elucidating the relationships between neural circuits and function using neurogenetic methods. PMID:25396742

  14. A Hypothetical Model of Crossing Bombyx mori Nucleopolyhedrovirus through Its Host Midgut Physical Barrier

    PubMed Central

    Cheng, Yang; Wang, Xue-Yang; Hu, Hao; Killiny, Nabil; Xu, Jia-Ping

    2014-01-01

    Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen of silkworm (B. mori) that causes severe economic losses each year. However, the molecular mechanisms of silkworm-BmNPV interactions, especially the silkworm proteins that can interact with the virus, are still largely unknown. In this study, the total and membrane proteins of silkworm midguts were displayed using one- and two-dimensional electrophoresis. A virus overlay assay was used to detect B. mori proteins that specifically bind to BmNPV particles. Twelve proteins were located and identified using mass spectrometry, and the different expression of the corresponding genes in BmNPV susceptible and resistant silkworm strains also indicated their involvement in BmNPV infection. The 12 proteins are grouped based on their potential roles in viral infection, for example, endocytosis, intracellular transportation, and host responses. Based on these results, we hypothesize the following: I) vacuolar ATP synthase catalytic subunit A and subunit B may be implicated in the process of the membrane fusion of virus and the release of the nucleocapsid into cytoplasm; II) actin, enolase and phosphoglycerate kinase are cytoskeleton associated proteins and may play an important role in BmNPV intracellular transportation; III) mitochondrial prohibitin complex protein 2, ganglioside-induced differentiation-associated protein, calreticulin, regucalcin-like isoform X1 and 60 kDa heat shock protein are involved in cell apoptosis regulation during BmNPV infection in larvae midguts; IV) ribosomal P0 may be associated with BmNPV infection by regulating gene expression of BmNPV; V) arginine kinase has a role in the antiviral activities against BmNPV. Our work should prove informative by providing multiple protein targets and a novel direction to investigate the molecular mechanisms of the interactions between silkworms and BmNPV. PMID:25502928

  15. Rheology and electrospinning of regenerated bombyx mori silk fibroin aqueous solutions.

    PubMed

    Hodgkinson, Tom; Chen, Ying; Bayat, Ardeshir; Yuan, Xue-Feng

    2014-04-14

    Bombyx mori silk fibroin (BMSF) has received considerable research interest as a potential biomaterial owing to its excellent mechanical properties and benign, versatile material fabrication options, including electrospinning. Despite this, characterizations of regenerated BMSF aqueous solutions and electrospun materials resulting from them are still very limited in the literature. This report details the rheological characterization of regenerated aqueous BMSF solutions under shear and elongational deformation. Well-characterized regenerated BMSF solutions were then systematically electrospun over a range of concentrations and process parameters to determine their effects on electrospinning processing windows and fiber morphology. BMSF solutions could not be electrospun successfully if BMSF concentration was below 20 wt % or the relaxation time measured using the CaBER rheometer was below 0.001 s. Electrospun BMSF fiber diameter was found to increase with solution concentration when stable electrospinning was achieved. An upper threshold of 30 wt % BMSF solution was identified for the formation of fibers with a circular cross section. Adding small amount of high molecular weight poly(ethylene oxide) was an effective rheological modifier that greatly improved the electrospinnability of BMSF solutions. Electrospinning BMSF-PEO solutions over a range of parameters significantly altered the fiber products. Increasing voltage from 0.5 to 1 kV/cm was found to decrease fiber diameter by approximately 50% (p < 0.001). Flow rate was found to have a significant effect on fiber diameter, which decreased with spinneret height. The results presented here provide valuable guidance in the production of BMSF electrospun materials with specific properties for tissue engineering and regenerative medicine.

  16. Functional Material Features of Bombyx mori Silk Light vs. Heavy Chain Proteins

    PubMed Central

    Zafar, Muhammad S.; Belton, David J.; Hanby, Benjamin; Kaplan, David L.; Perry, Carole C.

    2016-01-01

    Bombyx mori (BM) silk fibroin is composed of two different subunits; heavy chain and light chain fibroin linked by a covalent disulphide bond. Current methods of separating the two silk fractions is complicated and produces inadequate quantities of the isolated components for the study of the individual light and heavy chain silks with respect to new materials. We report a simple method of separating silk fractions using formic acid. The formic acid treatment partially releases predominately the light chain fragment (soluble fraction) and then the soluble fraction and insoluble fractions can be converted into new materials. The regenerated original (total) silk fibroin and the separated fractions (soluble vs. insoluble) had different molecular weights and showed distinctive pH stabilities against aggregation/precipitation based on particle charging. All silk fractions could be electrospun to give fibre mats with viscosity of the regenerated fractions being the controlling factor for successful electrospinning. The silk fractions could be mixed to give blends with different proportions of the two fractions to modify the diameter and uniformity of the electrospun fibres formed. The soluble fraction containing the light chain was able to modify the viscosity by thinning the insoluble fraction containing heavy chain fragments, perhaps analogous to its role in natural fibre formation where the light chain provides increased mobility and the heavy chain producing shear thickening effects. The simplicity of this new separation method should enable access to these different silk protein fractions and accelerate the identification of methods, modifications and potential applications of these materials in biomedical and industrial applications. PMID:25565556

  17. In silico identification of BESS-DC genes and expression analysis in the silkworm, Bombyx mori.

    PubMed

    Rao, Zhongchen; Duan, Jun; Xia, Qingyou; Feng, Qili

    2016-01-10

    BESS domain is a protein binding domain that can interact each other or with other domains. In this study, 323 BESS domain containing (BESS-DC) proteins were identified in 3328 proteomes. These BESS-DC genes pertain to 41 species of five phyla, most of which are arthropod insects. A BESS domain contains two α-helixes linked by a coil or β-turn. Phylogenetic tree and architecture analysis show that the BESS domain seems to generate along with the DNA-binding MADF domain. Two hundred thirty three BESS-DC genes (71.1%) contain at least one MADF domain, while 59 genes (18.2%) had only the BESS domain. In addition to BESS and MADF domains, some of genes also contain other ligand binding domains, such as DAO, DUS and NAD_C. Nineteen genes (5.8%) are associated with other DNA binding domains, such as Myb and BED. The BESS-DC genes can be divided into 17 subfamilies, eight of which have more than one clade. In Bombyx mori, 12 BESS-DC genes that do not contain intron in the BESS domain region were localized to eight chromosomes. Real-time PCR results showed that most of the B. mori BESS-DC genes highly expressed from late larval stage to adult stage. The results of sequence comparison and evolution analyses suggest a hypothesis that the BESS-DC genes may play a role in central nervous system development, long term memory and metamorphosis of insects of different phyla. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Effects of BmCPV Infection on Silkworm Bombyx mori Intestinal Bacteria.

    PubMed

    Sun, Zhenli; Lu, Yahong; Zhang, Hao; Kumar, Dhiraj; Liu, Bo; Gong, Yongchang; Zhu, Min; Zhu, Liyuan; Liang, Zi; Kuang, Sulan; Chen, Fei; Hu, Xiaolong; Cao, Guangli; Xue, Renyu; Gong, Chengliang

    2016-01-01

    The gut microbiota has a crucial role in the growth, development and environmental adaptation in the host insect. The objective of our work was to investigate the microbiota of the healthy silkworm Bombyx mori gut and changes after the infection of B. mori cypovirus (BmCPV). Intestinal contents of the infected and healthy larvae of B. mori of fifth instar were collected at 24, 72 and 144 h post infection with BmCPV. The gut bacteria were analyzed by pyrosequencing of the 16S rRNA gene. 147(135) and 113(103) genera were found in the gut content of the healthy control female (male) larvae and BmCPV-infected female (male) larvae, respectively. In general, the microbial communities in the gut content of healthy larvae were dominated by Enterococcus, Delftia, Pelomonas, Ralstonia and Staphylococcus, however the abundance change of each genus was depended on the developmental stage and gender. Microbial diversity reached minimum at 144 h of fifth instar larvae. The abundance of Enterococcus in the females was substantially lower and the abundance of Delftia, Aurantimonas and Staphylococcus was substantially higher compared to the males. Bacterial diversity in the intestinal contents decreased after post infection with BmCPV, whereas the abundance of both Enterococcus and Staphylococcus which belongs to Gram-positive were increased. Therefore, our findings suggested that observed changes in relative abundance was related to the immune response of silkworm to BmCPV infection. Relevance analysis of plenty of the predominant genera showed the abundance of the Enterococcus genus was in negative correlation with the abundance of the most predominant genera. These results provided insight into the relationship between the gut microbiota and development of the BmCPV-infected silkworm.

  19. Mechanism of enhanced Bombyx mori nucleopolyhedrovirus-resistance by titanium dioxide nanoparticles in silkworm.

    PubMed

    Xu, Kaizun; Li, Fanchi; Ma, Lie; Wang, Binbin; Zhang, Hua; Ni, Min; Hong, Fashui; Shen, Weide; Li, Bing

    2015-01-01

    The infection of Bombyx mori nucleopolyhedrovirus (BmNPV) in silkworms is often lethal. It is difficult to prevent, and its lethality is correlated with both viral particle characteristics and silkworm strains. Low doses of titanium dioxide nanoparticles (TiO2 NPs) can promote silkworm growth and improve its resistance to organophosphate pesticides. In this study, TiO2 NPs' effect on BmNPV resistance was investigated by analyzing the characteristics of BmNPV proliferation and transcriptional differences in silkworm midgut and the transcriptional changes of immunity related genes after feeding with TiO2 NPs. We found that low doses of TiO2 NPs improved the resistance of silkworm against BmNPV by 14.88-fold, with the mortalities of the experimental group and control group being 0.56% and 8.33% at 144 h, respectively. The proliferation of BmNPV in the midgut was significantly increased 72 h after infection in both experimental and control groups; the control group reached the peak at 120 h, while the experimental group took 24 more hours to reach the maximal value that was 12.63 times lower than the control, indicating that TiO2 NPs can inhibit BmNPV proliferation in the midgut. Consistently, the expression of the BmNPV-resistant gene Bmlipase-1 had the same increase pattern as the proliferation changes. Immune signaling pathway analysis revealed that TiO2 NPs inhibited the proliferation of silkworm BmNPV to reduce the activation levels of janus kinase/signal transducer and activator of transcription (JAK/STAT) and phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, while promoting the expression of Bmakt to improve the immunity. Overall, our results demonstrate that TiO2 NPs increase silkworm resistance against BmNPV by inhibiting virus proliferation and improving immunity in silkworms.

  20. Nutrigenetic Screening Strains of the Mulberry Silkworm, Bombyx mori, for Nutritional Efficiency

    PubMed Central

    Chinnaswamy, Ramesha; Lakshmi, Hothur; Kumari, Savarapu S.; Anuradha, Chebba M.; Kumar, Chitta S.

    2012-01-01

    The activity of sericulture is declining due the reduction of mulberry production area in sericulture practicing countries lead to adverse effects on silkworm rearing and cocoon production. Screening for nutrigenetic traits in silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) is an essential prerequisite for better understanding and development of nutritionally efficient breeds/hybrids, which show less food consumption with higher efficiency conversion. The aim of this study was to identify nutritionally efficient polyvoltine silkworm strains using the germplasm breeds RMW2, RMW3, RMW4, RMG3, RMG1, RMG4, RMG5, RMG6 and APM1 as the control. The 1st day of 5th stage silkworm larvae of polyvoltine strains were subjected to standard gravimetric analysis until spinning for three consecutive generations covering three different seasons on 19 nutrigenetic traits. Highly significant (p ≤ 0.001) differences were found among all nutrigenetic traits of polyvoltine silkworm strains in the experimental groups. The nutritionally efficient polvoltine silkworm strains were resulted by utilizing nutrition consumption index and efficiency of conversion of ingesta/cocoon traits as the index. Higher nutritional efficiency conversions were found in the polyvoltine silkworm strains on efficiency of conversion of ingesta to cocoon and shell than control. Comparatively smaller consumption index, respiration, metabolic rate with superior relative growth rate, and quantum of food ingesta and digesta requisite per gram of cocoon and shell were shown; the lowest amount was in new polyvoltine strains compared to the control. Furthermore, based on the overall nutrigenetic traits utilized as index or ‘biomarkers’, three polyvoltine silkworm strains (RMG4, RMW2, and RMW3) were identified as having the potential for nutrition efficiency conversion. The data from the present study advances our knowledge for the development of nutritionally efficient silkworm breeds/hybrids and their

  1. TRANSCRIPTION FACTOR Bmsage PLAYS A CRUCIAL ROLE IN SILK GLAND GENERATION IN SILKWORM, Bombyx mori.

    PubMed

    Xin, Hu-hu; Zhang, Deng-pan; Chen, Rui-ting; Cai, Zi-zheng; Lu, Yan; Liang, Shuang; Miao, Yun-gen

    2015-10-01

    Salivary gland secretion is altered in Drosophila embryos with loss of function of the sage gene. Saliva has a reduced volume and an increased electron density according to transmission electron microscopy, resulting in regions of tube dilation and constriction with intermittent tube closure. However, the precise functions of Bmsage in silkworm (Bombyx mori) are unknown, although its sequence had been deposited in SilkDB. From this, Bmsage is inferred to be a transcription factor that regulates the synthesis of silk fibroin and interacts with another silk gland-specific transcription factor, namely, silk gland factor-1. In this study, we introduced a germline mutation of Bmsage using the Cas9/sgRNA system, a genome-editing technology, resulting in deletion of Bmsage from the genome of B. mori. Of the 15 tested samples, seven displayed alterations at the target site. The mutagenesis efficiency was about 46.7% and there were no obvious off-target effects. In the screened homozygous mutants, silk glands developed poorly and the middle and posterior silk glands (MSG and PSG) were absent, which was significantly different from the wild type. The offspring of G0 mosaic silkworms had indel mutations causing 2- or 9-bp deletions at the target site, but exhibited the same abnormal silk gland structure. Mutant larvae containing different open-reading frames of Bmsage had the same silk gland phenotype. This illustrated that the mutant phenotype was due to Bmsage knockout. We conclude that Bmsage participates in embryonic development of the silk gland. © 2015 Wiley Periodicals, Inc.

  2. Intestinal microecology associated with fluoride resistance capability of the silkworm (Bombyx mori L.).

    PubMed

    Li, Guan-Nan; Xia, Xue-Juan; Tang, Wen-Chao; Zhu, Yong

    2016-08-01

    The silkworm (Bombyx mori L.) is an ideal model of Lepidoptera. However, the diversity and function of the intestinal microbiota in the gut of silkworm remain largely unknown. Changes in the intestinal microecology in fluoride-resistant strain T6 and fluoride-susceptible strain 734 of the silkworm in response to fluoride exposure were investigated. T6 and 734 were treated with 200 mg/kg fluoride (designated as T6-T and 734-T groups) and deionized water (designated as T6-C and 734-C groups). Culture-dependent approach revealed that the numbers of intestinal bacteria in the 734-T group significantly decreased compared with that in the 734-C group (4.8 ± 0.6 × 10(7) CFU/mL vs. 7.5 ± 0.7 × 10(7) CFU/mL; P < 0.05). Analyses of the intestinal content pH showed that the pH decreased in the 734-T group only. Additionally, SCFA concentrations significantly decreased in both treatment groups compared with the control groups. High-throughput sequencing indicated that the intestinal microbiota in the 734-T group was significantly more diverse than those in the other groups. The bacterial community was composed of two dominant groups (Firmicutes and Proteobacteria). Principal component analyses revealed a significant difference in the composition of the intestinal microbiota in the 734-T group compared with those in the other groups. Thaumarchaeota and Euryarchaeota were more abundant in the 734-T group, but they were less abundant in the other groups. This study enhances our understanding about the diversity and function of silkworm intestinal microbiota in response to fluoride exposure among silkworm strains with diverse resistance.

  3. Promoter analysis and RNA interference of CYP6ab4 in the silkworm Bombyx mori.

    PubMed

    Zhao, Guo-Dong; Zhang, Yi-Ling; Liu, Yun-Lei; Li, Bing; Chen, Yu-Hua; Xu, Ya-Xiang; Xia, Qing-You; Shen, Wei-De; Wei, Zheng-Guo

    2015-10-01

    In insects, cytochrome P450 monooxygenases (P450s) are involved in the metabolism of endogenous compounds such as steroid hormones and lipids. In this study, we measured the 20-hydroxyecdysone (20E)-induced transcriptional level of the CYP6ab4 gene using reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) with a dual spike-in strategy. We then probed possible physiological functions using RNAi experiments in the silkworm Bombyx mori. The activity of the CYP6ab4 promoter in various silkworm tissues was measured by firefly luciferase activity and normalized by Renilla luciferase activity. Our results showed that the activity of the CYP6ab4 promoter was highest in the malpighian tubule, followed by the fat body, the silk gland, the midgut, the epidermis, and the hemocyte. The essential region for basal and 20E-induced transcriptional activity was between -908 and -456 bp from the transcription start site. Through promoter truncation analysis using a dual-luciferase reporter assay in B. mori ovary cells (BmN), we showed that the region between -827 and -722 bp was essential for basal and 20E-induced transcriptional activity. Sequence analysis of this region revealed several potential transcriptional regulatory elements such as Hunchback (Hb) and BR-C Z. Mutation of the core bases of the BR-C Z binding site demonstrated that BR-C Z induces 20E-mediated CYP6ab4 transcription. Further identification of cis- and trans-elements and their roles in the upregulation of CYP6ab4 may be useful for elucidating the contribution of P450 to the response mechanism to 20E.

  4. Cloning and analysis of DnaJ family members in the silkworm, Bombyx mori.

    PubMed

    Li, Yinü; Bu, Cuiyu; Li, Tiantian; Wang, Shibao; Jiang, Feng; Yi, Yongzhu; Yang, Huipeng; Zhang, Zhifang

    2016-01-15

    Heat shock proteins (Hsps) are involved in a variety of critical biological functions, including protein folding, degradation, and translocation and macromolecule assembly, act as molecular chaperones during periods of stress by binding to other proteins. Using expressed sequence tag (EST) and silkworm (Bombyx mori) transcriptome databases, we identified 27 cDNA sequences encoding the conserved J domain, which is found in DnaJ-type Hsps. Of the 27 J domain-containing sequences, 25 were complete cDNA sequences. We divided them into three types according to the number and presence of conserved domains. By analyzing the gene structures, intron numbers, and conserved domains and constructing a phylogenetic tree, we found that the DnaJ family had undergone convergent evolution, obtaining new domains to expand the diversity of its family members. The acquisition of the new DnaJ domains most likely occurred prior to the evolutionary divergence of prokaryotes and eukaryotes. The expression of DnaJ genes in the silkworm was generally higher in the fat body. The tissue distribution of DnaJ1 proteins was detected by western blotting, demonstrating that in the fifth-instar larvae, the DnaJ1 proteins were expressed at their highest levels in hemocytes, followed by the fat body and head. We also found that the DnaJ1 transcripts were likely differentially translated in different tissues. Using immunofluorescence cytochemistry, we revealed that in the blood cells, DnaJ1 was mainly localized in the cytoplasm. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Genome-wide transcriptional response of silkworm (Bombyx mori) to infection by the microsporidian Nosema bombycis.

    PubMed

    Ma, Zhengang; Li, Chunfeng; Pan, Guoqing; Li, Zhihong; Han, Bing; Xu, Jinshan; Lan, Xiqian; Chen, Jie; Yang, Donglin; Chen, Quanmei; Sang, Qi; Ji, Xiaocun; Li, Tian; Long, Mengxian; Zhou, Zeyang

    2013-01-01

    Microsporidia have attracted much attention because they infect a variety of species ranging from protists to mammals, including immunocompromised patients with AIDS or cancer. Aside from the study on Nosema ceranae, few works have focused on elucidating the mechanism in host response to microsporidia infection. Nosema bombycis is a pathogen of silkworm pébrine that causes great economic losses to the silkworm industry. Detailed understanding of the host (Bombyx mori) response to infection by N. bombycis is helpful for prevention of this disease. A genome-wide survey of the gene expression profile at 2, 4, 6 and 8 days post-infection by N. bombycis was performed and results showed that 64, 244, 1,328, 1,887 genes were induced, respectively. Up to 124 genes, which are involved in basal metabolism pathways, were modulated. Notably, B. mori genes that play a role in juvenile hormone synthesis and metabolism pathways were induced, suggesting that the host may accumulate JH as a response to infection. Interestingly, N. bombycis can inhibit the silkworm serine protease cascade melanization pathway in hemolymph, which may be due to the secretion of serpins in the microsporidia. N. bombycis also induced up-regulation of several cellular immune factors, in which CTL11 has been suggested to be involved in both spore recognition and immune signal transduction. Microarray and real-time PCR analysis indicated the activation of silkworm Toll and JAK/STAT pathways. The notable up-regulation of antimicrobial peptides, including gloverins, lebocins and moricins, strongly indicated that antimicrobial peptide defense mechanisms were triggered to resist the invasive microsporidia. An analysis of N. bombycis-specific response factors suggested their important roles in anti-microsporidia defense. Overall, this study primarily provides insight into the potential molecular mechanisms for the host-parasite interaction between B. mori and N. bombycis and may provide a foundation for

  6. Genome-Wide Transcriptional Response of Silkworm (Bombyx mori) to Infection by the Microsporidian Nosema bombycis

    PubMed Central

    Pan, Guoqing; Li, Zhihong; Han, Bing; Xu, Jinshan; Lan, Xiqian; Chen, Jie; Yang, Donglin; Chen, Quanmei; Sang, Qi; Ji, Xiaocun; Li, Tian; Long, Mengxian; Zhou, Zeyang

    2013-01-01

    Microsporidia have attracted much attention because they infect a variety of species ranging from protists to mammals, including immunocompromised patients with AIDS or cancer. Aside from the study on Nosema ceranae, few works have focused on elucidating the mechanism in host response to microsporidia infection. Nosema bombycis is a pathogen of silkworm pébrine that causes great economic losses to the silkworm industry. Detailed understanding of the host (Bombyx mori) response to infection by N. bombycis is helpful for prevention of this disease. A genome-wide survey of the gene expression profile at 2, 4, 6 and 8 days post-infection by N. bombycis was performed and results showed that 64, 244, 1,328, 1,887 genes were induced, respectively. Up to 124 genes, which are involved in basal metabolism pathways, were modulated. Notably, B. mori genes that play a role in juvenile hormone synthesis and metabolism pathways were induced, suggesting that the host may accumulate JH as a response to infection. Interestingly, N. bombycis can inhibit the silkworm serine protease cascade melanization pathway in hemolymph, which may be due to the secretion of serpins in the microsporidia. N. bombycis also induced up-regulation of several cellular immune factors, in which CTL11 has been suggested to be involved in both spore recognition and immune signal transduction. Microarray and real-time PCR analysis indicated the activation of silkworm Toll and JAK/STAT pathways. The notable up-regulation of antimicrobial peptides, including gloverins, lebocins and moricins, strongly indicated that antimicrobial peptide defense mechanisms were triggered to resist the invasive microsporidia. An analysis of N. bombycis-specific response factors suggested their important roles in anti-microsporidia defense. Overall, this study primarily provides insight into the potential molecular mechanisms for the host-parasite interaction between B. mori and N. bombycis and may provide a foundation for

  7. Nutrigenetic Screening Strains of the Mulberry Silkworm, Bombyx mori, for Nutritional Efficiency

    PubMed Central

    Ramesha, Chinnaswamy; Lakshmi, Hothur; Kumari, Savarapu Sugnana; Anuradha, Chevva M.; Kumar, Chitta Suresh

    2012-01-01

    The activity of sericulture is declining due the reduction of mulberry production area in sericulture practicing countries lead to adverse effects on silkworm rearing and cocoon production. Screening for nutrigenetic traits in silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) is an essential prerequisite for better understanding and development of nutritionally efficient breeds/hybrids, which show less food consumption with higher efficiency conversion. The aim of this study was to identify nutritionally efficient polyvoltine silkworm strains using the germplasm breeds RMW2, RMW3, RMW4, RMG3, RMG1, RMG4, RMG5, RMG6 and APM1 as the control. The 1st day of 5th stage silkworm larvae of polyvoltine strains were subjected to standard gravimetric analysis until spinning for three consecutive generations covering 3 different seasons on 19 nutrigenetic traits. Highly significant (p ≤ 0.001) differences were found among all nutrigenetic traits of polyvoltine silkworm strains in the experimental groups. The nutritionally efficient polvoltine silkworm strains were resulted by utilizing nutrition consumption index and efficiency of conversion of ingesta/cocoon traits as the index. Higher nutritional efficiency conversions were found in the polyvoltine silkworm strains on efficiency of conversion of ingesta to cocoon and shell than control. Comparatively smaller consumption index, respiration, metabolic rate with superior relative growth rate, and quantum of food ingesta and digesta requisite per gram of cocoon and shell were found; the lowest amount was in new polyvoltine strains compared to the control. Furthermore, based on the overall nutrigenetic traits utilized as index or ‘biomarkers’, three polyvoltine silkworm strains (RMG4, RMW2, and RMW3) were identified as having the potential for nutrition efficiency conversion. The data from the present study advances our knowledge for the development of nutritionally efficient silkworm breeds/hybrids and their effective

  8. Precocious metamorphosis in the juvenile hormone-deficient mutant of the silkworm, Bombyx mori.

    PubMed

    Daimon, Takaaki; Kozaki, Toshinori; Niwa, Ryusuke; Kobayashi, Isao; Furuta, Kenjiro; Namiki, Toshiki; Uchino, Keiro; Banno, Yutaka; Katsuma, Susumu; Tamura, Toshiki; Mita, Kazuei; Sezutsu, Hideki; Nakayama, Masayoshi; Itoyama, Kyo; Shimada, Toru; Shinoda, Tetsuro

    2012-01-01

    Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several "moltinism" mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval-larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval-pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH-deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis.

  9. Nutrigenetic screening strains of the mulberry silkworm, Bombyx mori, for nutritional efficiency.

    PubMed

    Chinnaswamy, Ramesha; Lakshmi, Hothur; Kumari, Savarapu S; Anuradha, Chebba M; Kumar, Chitta S

    2012-01-01

    The activity of sericulture is declining due the reduction of mulberry production area in sericulture practicing countries lead to adverse effects on silkworm rearing and cocoon production. Screening for nutrigenetic traits in silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) is an essential prerequisite for better understanding and development of nutritionally efficient breeds/hybrids, which show less food consumption with higher efficiency conversion. The aim of this study was to identify nutritionally efficient polyvoltine silkworm strains using the germplasm breeds RMW(2), RMW(3), RMW(4), RMG(3), RMG(1), RMG(4), RMG(5), RMG(6) and APM(1) as the control. The 1(st) day of 5(th) stage silkworm larvae of polyvoltine strains were subjected to standard gravimetric analysis until spinning for three consecutive generations covering three different seasons on 19 nutrigenetic traits. Highly significant (p ≤ 0.001) differences were found among all nutrigenetic traits of polyvoltine silkworm strains in the experimental groups. The nutritionally efficient polvoltine silkworm strains were resulted by utilizing nutrition consumption index and efficiency of conversion of ingesta/cocoon traits as the index. Higher nutritional efficiency conversions were found in the polyvoltine silkworm strains on efficiency of conversion of ingesta to cocoon and shell than control. Comparatively smaller consumption index, respiration, metabolic rate with superior relative growth rate, and quantum of food ingesta and digesta requisite per gram of cocoon and shell were shown; the lowest amount was in new polyvoltine strains compared to the control. Furthermore, based on the overall nutrigenetic traits utilized as index or 'biomarkers', three polyvoltine silkworm strains (RMG(4), RMW(2), and RMW(3)) were identified as having the potential for nutrition efficiency conversion. The data from the present study advances our knowledge for the development of nutritionally efficient silkworm breeds

  10. Transgenic characterization of two testis-specific promoters in the silkworm, Bombyx mori.

    PubMed

    Xu, J; Bi, H; Chen, R; Aslam, A F M; Li, Z; Ling, L; Zeng, B; Huang, Y; Tan, A

    2015-04-01

    Sex-specific regulatory elements are key components for developing insect genetic sexing systems. The current insect genetic sexing system mainly uses a female-specific modification system whereas little success was reported on male-specific genetic modification. In the silkworm Bombyx mori, a lepidopteran model insect with economic importance, a transgene-based, female-specific lethality system has been established based on sex-specific alternative splicing factors and a female-specific promoter BmVgp (vitellogenin promoter) has been identified. However, no male-specific regulatory elements have yet been identified. Here we report the transgenic identification of two promoters that drive reporter gene expression in a testis-specific manner in B. mori. Putative promoter sequences from the B. mori Radial spoke head 1 gene (BmR1) and beta-tubulin 4 gene (Bmβ4) were introduced using piggybac-based germline transformation. In transgenic silkworms, expression of the reporter gene enhanced green fluorescent protein (EGFP) directed by either BmR1 promoter (BmR1p) or Bmβ4p showed precisely testis-specific manners from the larval to adult stage. Furthermore, EGFP expression of these two transgenic lines showed different localization in the testis, indicating that BmR1p or Bmβ4p might be used as distinct regulatory elements in directing testis-specific gene expression. Identification of these testis-specific promoters not only contributes to a better understanding of testis-specific gene function in insects, but also has potential applications in sterile insect techniques for pest management.

  11. A new arylalkylamine N-acetyltransferase in silkworm (Bombyx mori) affects integument pigmentation.

    PubMed

    Long, Yaohang; Li, Jiaorong; Zhao, Tianfu; Li, Guannan; Zhu, Yong

    2015-04-01

    Dopamine is a precursor for melanin synthesis. Arylalkylamine N-acetyltransferase (AANAT) is involved in the melatonin formation in insects because it could catalyze the transformation from dopamine to dopamine-N-acetyldopamine. In this study, we identified a new AANAT gene in the silkworm (Bombyx mori) and assessed its role in the silkworm. The cDNA of this gene encodes 233 amino acids that shares 57 % amino acid identity with the Bm-iAANAT protein. We thus refer to this gene as Bm-iAANAT2. To investigate the role of Bm-iAANAT2, we constructed a transgenic interference system using a 3xp3 promoter to suppress the expression of Bm-iAANAT2 in the silkworm. We observed that melanin deposition occurs in the head and integument in transgenic lines. To verify the melanism pattern, dopamine content and the enzyme activity of AANAT were determined by high-performance liquid chromatography (HPLC). We found that an increase in dopamine levels affects melanism patterns on the heads of transgenic B. mori. A reduction in the enzyme activity of AANAT leads to changes in dopamine levels. We analyzed the expression of the Bm-iAANAT2 genes by qPCR and found that the expression of Bm-iAANAT2 gene is significantly lower in transgenic lines. Our results lead us to conclude that Bm-iAANAT2 is a new arylalkylamine N-acetyltransferase gene in the silkworm and is involved in the metabolism of the dopamine to avoid the generation of melanin.

  12. Identification of two juvenile hormone inducible transcription factors from the silkworm, Bombyx mori.

    PubMed

    Matsumoto, Hitoshi; Ueno, Chihiro; Nakamura, Yuki; Kinjoh, Terunori; Ito, Yuka; Shimura, Sachiko; Noda, Hiroaki; Imanishi, Shigeo; Mita, Kazuei; Fujiwara, Haruhiko; Hiruma, Kiyoshi; Shinoda, Tetsuro; Kamimura, Manabu

    2015-09-01

    Juvenile hormone (JH) regulates many physiological processes in insects. However, the signal cascades in which JH is active have not yet been fully elucidated, particularly in comparison to another major hormone ecdysteroid. Here we identified two JH inducible transcription factors as candidate components of JH signaling pathways in the silkworm, Bombyx mori. DNA microarray analysis showed that expression of two transcription factor genes, E75 and Enhancer of split mβ (E(spl)mβ), was induced by juvenile hormone I (JH I) in NIAS-Bm-aff3 cells. Real time RT-PCR analysis confirmed that expression of four E75 isoforms (E75A, E75B, E75C and E75D) and E(spl)mβ was 3-8 times greater after JH I addition. Addition of the protein synthesis inhibitor cycloheximide did not suppress JH-induced expression of the genes, indicating that they were directly induced by JH. JH-induced expression of E75 and E(spl)mβ was also observed in four other B. mori cell lines and in larval hemocytes of final instar larvae. Notably, E75A expression was induced very strongly in larval hemocytes by topical application of the JH analog fenoxycarb; the level of induced expression was comparable to that produced by feeding larvae with 20-hydroxyecdysone. These results suggest that E75 and E(spl)mβ are general and direct target genes of JH and that the transcription factors encoded by these genes play important roles in JH signaling.

  13. Nutrigenetic screening strains of the mulberry silkworm, Bombyx mori, for nutritional efficiency.

    PubMed

    Ramesha, Chinnaswamy; Lakshmi, Hothur; Kumari, Savarapu Sugnana; Anuradha, Chevva M; Kumar, Chitta Suresh

    2012-01-01

    The activity of sericulture is declining due the reduction of mulberry production area in sericulture practicing countries lead to adverse effects on silkworm rearing and cocoon production. Screening for nutrigenetic traits in silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) is an essential prerequisite for better understanding and development of nutritionally efficient breeds/hybrids, which show less food consumption with higher efficiency conversion. The aim of this study was to identify nutritionally efficient polyvoltine silkworm strains using the germplasm breeds RMW(2), RMW(3), RMW(4), RMG(3), RMG(1), RMG(4), RMG(5), RMG(6) and APM(1) as the control. The 1(st) day of 5(th) stage silkworm larvae of polyvoltine strains were subjected to standard gravimetric analysis until spinning for three consecutive generations covering 3 different seasons on 19 nutrigenetic traits. Highly significant (p ≤ 0.001) differences were found among all nutrigenetic traits of polyvoltine silkworm strains in the experimental groups. The nutritionally efficient polvoltine silkworm strains were resulted by utilizing nutrition consumption index and efficiency of conversion of ingesta/cocoon traits as the index. Higher nutritional efficiency conversions were found in the polyvoltine silkworm strains on efficiency of conversion of ingesta to cocoon and shell than control. Comparatively smaller consumption index, respiration, metabolic rate with superior relative growth rate, and quantum of food ingesta and digesta requisite per gram of cocoon and shell were found; the lowest amount was in new polyvoltine strains compared to the control. Furthermore, based on the overall nutrigenetic traits utilized as index or 'biomarkers', three polyvoltine silkworm strains (RMG(4), RMW(2), and RMW(3)) were identified as having the potential for nutrition efficiency conversion. The data from the present study advances our knowledge for the development of nutritionally efficient silkworm breeds

  14. Depletion of juvenile hormone esterase extends larval growth in Bombyx mori.

    PubMed

    Zhang, Zhongjie; Liu, Xiaojing; Shiotsuki, Takahiro; Wang, Zhisheng; Xu, Xia; Huang, Yongping; Li, Muwang; Li, Kai; Tan, Anjiang

    2017-02-01

    Two major hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), regulate insect growth and development according to their precisely coordinated titres, which are controlled by both biosynthesis and degradation pathways. Juvenile hormone esterase (JHE) is the primary JH-specific degradation enzyme that plays a key role in regulating JH titers, along with JH epoxide hydrolase (JHEH) and JH diol kinase (JHDK). In the current study, a loss-of-function analysis of JHE in the silkworm, Bombyx mori, was performed by targeted gene disruption using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system. Depletion of B. mori JHE (BmJHE) resulted in the extension of larval stages, especially the penultimate and ultimate larval stages, without deleterious effects to silkworm physiology. The expression of JHEH and JHDK was upregulated in mutant animals, indicating the existence of complementary routes in the JH metabolism pathway in which inactivation of one enzyme will activate other enzymes. RNA-Seq analysis of mutant animals revealed that genes involved in protein processing in the endoplasmic reticulum and in amino acid metabolism were affected by BmJHE depletion. Depletion of JHE and subsequent delayed JH metabolism activated genes in the TOR pathway, which are ultimately responsible for extending larval growth. The transgenic Cas9 system used in the current study provides a promising approach for analysing the actions of JH, especially in nondrosophilid insects. Furthermore, prolonging larval stages produced larger larvae and cocoons, which is greatly beneficial to silk production. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Bombyx mori cecropin A has a high antifungal activity to entomopathogenic fungus Beauveria bassiana.

    PubMed

    Lu, Dingding; Geng, Tao; Hou, Chengxiang; Huang, Yuxia; Qin, Guangxing; Guo, Xijie

    2016-05-25

    A cDNA encoding cecropin A (CecA) was cloned from the larvae of silkworm, Bombyx mori, using RT-PCR. It encodes a protein of 63 amino acids, containing a 22 amino acid signal peptide and a 37 amino acid mat peptide of functional domain. The CecA secondary structure contains two typical amphiphilic α-helices. Real-time qPCR analysis revealed that CecA was expressed in all the tissues tested, including cuticle, fat body, hemocytes, Malpighian tubule, midgut and silk gland in the silkworm larvae with the highest expression in the fat body and hemocytes. The gene expression of B. mori CecA was rapidly induced by Beauveria bassiana challenge and reached maximum levels at 36h after inoculation in third instar larvae. In the fifth instar larvae infected with B. bassiana, the relative expression level of CecA was upregulated in fat body and hemocytes, but not in cuticle, Malpighian tubule, midgut and silk gland. The cDNA segment of the CecA was inserted into the expression plasmid pET-30a(+) to construct a recombinant expression plasmid. Western blot results revealed that his-tagged fusion protein was successfully expressed and purified. Then the mat peptide of CecA was chemically synthesized with C-terminus amidation for in vivo antifungal assay and purity achieved 93.7%. Mass spectrometry and SDS-PAGE showed its molecular weight to be 4046.95Da. Antifungal assays indicated that the B. mori CecA had a high antifungal activity to entomopathogenic fungus B. bassiana both in vitro and in vivo in the silkworm larvae. This is the first report that the CecA is effective to inhibit B. bassiana inside the body of silkworm.

  16. A diversity of U1 small nuclear RNAs in the silk moth Bombyx mori.

    PubMed

    Sierra-Montes, J M; Pereira-Simon, S; Freund, A V; Ruiz, L M; Szmulewicz, M N; Herrera, R J

    2003-01-01

    Variants of U1 small nuclear RNAs (snRNAs) have been previously detected in a permanent cell line (BmN) of the silk moth Bombyx mori. In this study, the existence of U1 snRNA isoforms in the silk gland (SG) of the organism is investigated. The polyploidy (approximately 200,000X the 2N somatic value) state of the B. mori silk gland cells represents a unique system to explore the potential presence and differential expression of multiple U1 variants in a normal tissue. B. mori U1-specific RT-PCR libraries from the silk gland were generated and five U1 isoforms were isolated and characterized. Nucleotide differences, structural alterations, as well as protein and RNA interaction sites were examined in these variants and compared to the previously reported isoforms from the transformed BmN cell line. In all these SG U1 variants, variant sites and inter-species differences are located in moderately conserved regions. Substitutional or compensatory changes were found in the double stranded areas and clustered in moderately conserved regions. Some of the changes generate stronger base pairing. Calculated free energy (DeltaG) values for the entire U1 snRNA secondary structures and for the individual stem/loops (I, II, III and IV) domains of the isoforms were generated and compared to determine their structural stability. Using phylogenetic analysis, an evolutionary parallelism is observed between the polymorphic sites in B. mori and variant locations found among animal and plant species.

  17. Transcriptome analysis of the silkworm (Bombyx mori) by high-throughput RNA sequencing.

    PubMed

    Li, Yinü; Wang, Guozeng; Tian, Jian; Liu, Huifen; Yang, Huipeng; Yi, Yongzhu; Wang, Jinhui; Shi, Xiaofeng; Jiang, Feng; Yao, Bin; Zhang, Zhifang

    2012-01-01

    The domestic silkworm, Bombyx mori, is a model insect with important economic value for silk production that also acts as a bioreactor for biomaterial production. The functional complexity of the silkworm transcriptome has not yet been fully elucidated, although genomic sequencing and other tools have been widely used in its study. We explored the transcriptome of silkworm at different developmental stages using high-throughput paired-end RNA sequencing. A total of about 3.3 gigabases (Gb) of sequence was obtained, representing about a 7-fold coverage of the B. mori genome. From the reads that were mapped to the genome sequence; 23,461 transcripts were obtained, 5,428 of them were novel. Of the 14,623 predicted protein-coding genes in the silkworm genome database, 11,884 of them were found to be expressed in the silkworm transcriptome, giving a coverage of 81.3%. A total of 13,195 new exons were detected, of which, 5,911 were found in the annotated genes in the Silkworm Genome Database (SilkDB). An analysis of alternative splicing in the transcriptome revealed that 3,247 genes had undergone alternative splicing. To help with the data analysis, a transcriptome database that integrates our transcriptome data with the silkworm genome data was constructed and is publicly available at http://124.17.27.136/gbrowse2/. To our knowledge, this is the first study to elucidate the silkworm transcriptome using high-throughput RNA sequencing technology. Our data indicate that the transcriptome of silkworm is much more complex than previously anticipated. This work provides tools and resources for the identification of new functional elements and paves the way for future functional genomics studies.

  18. BmTGIF, a Bombyx mori Homolog of Drosophila DmTGIF, Regulates Progression of Spermatogenesis

    PubMed Central

    Sheng, Jie; Xue, Renyu; Gong, Chengliang

    2012-01-01

    TG-interacting factor (TGIF) in Drosophila consists of two tandemly-repeated genes, achintya (Dmachi) and vismay (Dmvis), which act as transcriptional activators in Drosophila spermatogenesis. In contrast, TGIF in humans is a transcriptional repressor that binds directly to DNA or interacts with corepressors to repress the transcription of target genes. In this study, we investigated the characteristics and functions of BmTGIF, a Bombyx mori homolog of DmTGIF. Like DmTGIF, BmTGIF is predominantly expressed in the testes and ovaries. Four alternatively spliced isoforms could be isolated from testes, and two isoforms from ovaries. Quantitative polymerase chain reaction indicated BmTGIF was abundantly expressed in the testis of 3rd instar larvae, when the testis is almost full of primary spermatocytes. The results of luciferase assays indicated that BmTGIF contains two adjacent acidic domains that activate the transcription of reporter genes. Immunofluorescence assay in BmN cells showed that the BmTGIF protein was located mainly in the nucleus, and paraffin sections of testis showed BmTGIF was grossly expressed in primary spermatocytes and mature sperms. Consistent with the role of DmVis in Drosophila development, BmTGIF significantly affected spermatid differentiation, as indicated by hematoxylin-eosin staining of paraffin sections of testis from BmTGIF-small interfering RNA (siRNA)-injected male silkworms. Co-immunoprecipitation experiments suggested that BmTGIF interacted with BmAly, and that they may recruit other factors to form a complex to regulate the genes required for meiotic divisions and spermatid differentiation. The results of this analysis of BmTGIF will improve our understanding of the mechanism of spermatid differentiation in B. mori, with potential applications for pest control. PMID:23152760

  19. Comparing the microstructure and mechanical properties of Bombyx mori and Antheraea pernyi cocoon composites.

    PubMed

    Guan, Juan; Zhu, Wenshu; Liu, Binghe; Yang, Kang; Vollrath, Fritz; Xu, Jun

    2017-01-01

    Silkworm cocoon material is a natural composite consisting of silk fibres and sericin glues. Both domestic and wild silkworms produce cocoons but with different functionality - one selected by man for textile manufacture whereas the other selected by Nature to provide damage-tolerant housing. To understand the structure--property relationship of cocoons, we evaluated and compared the microstructure and mechanical properties of two representative cocoon walls. It appears that a "brittle and weak" composite is produced by domestic Bombyx mori (B. mori) while a "tough and strong" composite is made by wild Antheraea pernyi (A. pernyi). The superior mechanical performance of A. pernyi cocoons can be attributed to both the material properties and the fibre network microstructures. Failure mechanisms and different failure modes for cocoon fibre composites were also proposed. A finite element model revealed qualitatively the effect of fibre properties and inter-fibre bonding strength on the mechanical properties of the fibre network. It emerged that both good mechanical properties of fibres and robust inter-fibre bonding were required for tough and strong fibre composites. The new insights could inspire new designs of synthetic fibre composites with enhanced mechanical properties. Natural cocoons are an important group of natural fibre composites with versatile functionalities. Previous studies have focused on the diversity of cocoon species and different morphological and mechanical features. It was suggested that the cocoon network structure determined the final mechanical properties of the cocoon composite. Nevertheless, the full structure-propertyfunction relationships for the cocoon composite are not understood. By studying two distinct cocoon species with specific functionalities, we prove that the mechanical properties of two cocoons are determined by both network properties and fibre properties. A robust fibre network is the prerequisite, within which the good

  20. Mechanism of Enhanced Bombyx mori Nucleopolyhedrovirus-Resistance by Titanium Dioxide Nanoparticles in Silkworm

    PubMed Central

    Zhang, Hua; Ni, Min; Hong, Fashui; Shen, Weide; Li, Bing

    2015-01-01

    The infection of Bombyx mori nucleopolyhedrovirus (BmNPV) in silkworms is often lethal. It is difficult to prevent, and its lethality is correlated with both viral particle characteristics and silkworm strains. Low doses of titanium dioxide nanoparticles (TiO2 NPs) can promote silkworm growth and improve its resistance to organophosphate pesticides. In this study, TiO2 NPs’ effect on BmNPV resistance was investigated by analyzing the characteristics of BmNPV proliferation and transcriptional differences in silkworm midgut and the transcriptional changes of immunity related genes after feeding with TiO2 NPs. We found that low doses of TiO2 NPs improved the resistance of silkworm against BmNPV by 14.88-fold, with the mortalities of the experimental group and control group being 0.56% and 8.33% at 144 h, respectively. The proliferation of BmNPV in the midgut was significantly increased 72 h after infection in both experimental and control groups; the control group reached the peak at 120 h, while the experimental group took 24 more hours to reach the maximal value that was 12.63 times lower than the control, indicating that TiO2 NPs can inhibit BmNPV proliferation in the midgut. Consistently, the expression of the BmNPV-resistant gene Bmlipase-1 had the same increase pattern as the proliferation changes. Immune signaling pathway analysis revealed that TiO2 NPs inhibited the proliferation of silkworm BmNPV to reduce the activation levels of janus kinase/signal transducer and activator of transcription (JAK/STAT) and phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, while promoting the expression of Bmakt to improve the immunity. Overall, our results demonstrate that TiO2 NPs increase silkworm resistance against BmNPV by inhibiting virus proliferation and improving immunity in silkworms. PMID:25692869

  1. Establishment of tools for neurogenetic analysis of sexual behavior in the silkmoth, Bombyx mori.

    PubMed

    Kiya, Taketoshi; Morishita, Koudai; Uchino, Keiro; Iwami, Masafumi; Sezutsu, Hideki

    2014-01-01

    Silkmoth, Bombyx mori, is an ideal model insect for investigating the neural mechanisms underlying sex pheromone-induced innate behavior. Although transgenic techniques and the GAL4/UAS system are well established in the silkmoth, genetic tools useful for investigating brain function at the neural circuit level have been lacking. In the present study, we established silkmoth strains in which we could visualize neural projections (UAS-mCD8GFP) and cell nucleus positions (UAS-GFP.nls), and manipulate neural excitability by thermal stimulation (UAS-dTrpA1). In these strains, neural projections and nucleus position were reliably labeled with green fluorescent protein in a GAL4-dependent manner. Further, the behavior of silkworm larvae and adults could be controlled by GAL4-dependent misexpression of dTrpA1. Ubiquitous dTrpA1 misexpression led both silkmoth larvae and adults to exhibit seizure-like phenotypes in a heat stimulation-dependent manner. Furthermore, dTrpA1 misexpression in the sex pheromone receptor neurons of male silkmoths allowed us to control male sexual behavior by changing the temperature. Thermally stimulated male silkmoths exhibited full sexual behavior, including wing-flapping, orientation, and attempted copulation, and precisely approached a thermal source in a manner similar to male silkmoths stimulated with the sex pheromone. These findings indicate that a thermogenetic approach using dTrpA1 is feasible in Lepidopteran insects and thermogenetic analysis of innate behavior is applicable in the silkmoth. These tools are essential for elucidating the relationships between neural circuits and function using neurogenetic methods.

  2. Proteomic analysis of BmN cell lipid rafts reveals roles in Bombyx mori nucleopolyhedrovirus infection.

    PubMed

    Hu, Xiaolong; Zhu, Min; Liang, Zi; Kumar, Dhiraj; Chen, Fei; Zhu, Liyuan; Kuang, Sulan; Xue, Renyu; Cao, Guangli; Gong, Chengliang

    2017-04-01

    The mechanism of how Bombyx mori nucleopolyhedrovirus (BmNPV) enters cells is unknown. The primary components of membrane lipid rafts are proteins and cholesterol, and membrane lipid rafts are thought to be an active region for host-viral interactions. However, whether they contribute to the entry of BmNPV into silkworm cells remains unclear. In this study, we explored the membrane protein components of lipid rafts from BmN cells with mass spectrometry (MS). Proteins and cholesterol were investigated after establishing infection with BmNPV in BmN cells. In total, 222 proteins were identified in the lipid rafts, and Gene Ontology (GO) annotation analysis showed that more than 10% of these proteins had binding and catalytic functions. We then identified proteins that potentially interact between lipid rafts and BmNPV virions using the Virus Overlay Protein Blot Assay (VOPBA). A total of 65 proteins were analyzed with MS, and 7 were predicted to be binding proteins involved in BmNPV cellular invasion, including actin, kinesin light chain-like isoform X2, annexin B13, heat-shock protein 90, barrier-to-autointegration factor B-like and serine/arginine-rich splicing factor 1 A-like. When the cholesterol of the lipid rafts from the membrane was depleted by methyl-β-cyclodextrin (MβCD), BmNPV entry into BmN cells was blocked. However, supplying cholesterol into the medium rescued the BmNPV infection ability. These results show that membrane lipid rafts may be the active regions for the entry of BmNPV into cells, and the components of membrane lipid rafts may be candidate targets for improving the resistance of the silkworm to BmNPV.

  3. The effect of bovine milk on the growth of Bombyx mori.

    PubMed

    Konala, Niharika; Abburi, Praveena; Bovilla, Venugopal Reddy; Mamillapalli, Anitha

    2013-01-01

    Bombyx mori L. (Lepidoptera: Bombycidae) is a well-studied Lepidopteran model system because of its morphology, life cycle, and economic importance. Many scientists have placed importance on enhancing the economic traits of B. mori because it's larvae, silkworms, are vital in the production of silk. In this study, the effect of bovine milk on B. mori growth was tested. Bovine milk contains several components that aid in healthy growth. The treatment was given to fifth instar B. mori larvae because the fifth instar period is when B. mori eats voraciously and shows maximum growth among all its larval stages. The larvae were treated with fresh mulberry, Morus L. (Rosales: Moraceae), leaves and mulberry leaves dipped in milk from the first day of the fifth instar. Treatments were given on alternate days, and the silkworms were weighed every day to determine whether milk had any role in enhancing the weight of the larvae. Cocoon weights were measured, as the weight indicates the approximate amount of silk that can be reeled. The results showed that larvae gained 82.5% more weight by the end of fifth instar larval when fed with mulberry leaves dipped in milk than when fed with fresh mulberry leaves without milk. The larvae fed with milk-treated leaves gained 310% weight from day 1 to day 7 of the fifth instar, while the larvae fed with fresh leaves gained 153% weight in the same timespan. In addition, cocoon weight increased by 8% when milk was added compared to when it was not. These results suggest that B. mori larvae can be fed mulberry leaves treated with bovine milk for better growth rate and increased silk production.

  4. Severe developmental timing defects in the prothoracicotropic hormone (PTTH)-deficient silkworm, Bombyx mori.

    PubMed

    Uchibori-Asano, Miwa; Kayukawa, Takumi; Sezutsu, Hideki; Shinoda, Tetsuro; Daimon, Takaaki

    2017-08-01

    The insect neuropeptide prothoracicotropic hormone (PTTH) triggers the biosynthesis and release of the molting hormone ecdysone in the prothoracic gland (PG), thereby controlling the timing of molting and metamorphosis. Despite the well-documented physiological role of PTTH and its signaling pathway in the PG, it is not clear whether PTTH is an essential hormone for ecdysone biosynthesis and development. To address this question, we established and characterized a PTTH knockout line in the silkworm, Bombyx mori. We found that PTTH knockouts showed a severe developmental delay in both the larval and pupal stages. Larval phenotypes of PTTH knockouts can be classified into three major classes: (i) developmental arrest during the second larval instar, (ii) precocious metamorphosis after the fourth larval instar (one instar earlier in comparison to the control strain), and (iii) metamorphosis to normal-sized pupae after completing the five larval instar stages. In PTTH knockout larvae, peak levels of ecdysone titers in the hemolymph were dramatically reduced and the timing of peaks was delayed, suggesting that protracted larval development is a result of the reduced and delayed synthesis of ecdysone in the PG. Despite these defects, low basal levels of ecdysone were maintained in PTTH knockout larvae, suggesting that the primary role of PTTH is to upregulate ecdysone biosynthesis in the PG during molting stages, and low basal levels of ecdysone can be maintained in the absence of PTTH. We also found that mRNA levels of genes involved in ecdysone biosynthesis and ecdysteroid signaling pathways were significantly reduced in PTTH knockouts. Our results provide genetic evidence that PTTH is not essential for development, but is required to coordinate growth and developmental timing. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Bombyx E75 isoforms display stage- and tissue-specific responses to 20-hydroxyecdysone

    PubMed Central

    Li, Kang; Guo, Enen; Hossain, Muktadir S.; Li, Qingrong; Cao, Yang; Tian, Ling; Deng, Xiaojuan; Li, Sheng

    2015-01-01

    Resulted from alternative splicing of the 5′ exons, the nuclear receptor gene E75 in the silkworm, Bombyx mori, processes three mRNA isoforms, BmE75A, BmE75B and BmE75C. From the early 5th larval instar to the prepupal stages, BmE75A mRNA and protein levels in the prothoracic glands display developmental profiles similar to ecdysteroid titer. In the fat body, mRNA levels but not protein levels of all three BmE75 isoforms correlate with ecdysteroid titer; moreover, proteins of all three BmE75 isoforms disappear at the prepupal stages, and a modified BmE75 protein with smaller molecular weight and cytoplasm localization occurs. At the early 5th larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E. At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues. In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level. PMID:26166384

  6. Precocious Metamorphosis in the Juvenile Hormone–Deficient Mutant of the Silkworm, Bombyx mori

    PubMed Central

    Daimon, Takaaki; Kozaki, Toshinori; Niwa, Ryusuke; Kobayashi, Isao; Furuta, Kenjiro; Namiki, Toshiki; Uchino, Keiro; Banno, Yutaka; Katsuma, Susumu; Tamura, Toshiki; Mita, Kazuei; Sezutsu, Hideki; Nakayama, Masayoshi; Itoyama, Kyo; Shimada, Toru; Shinoda, Tetsuro

    2012-01-01

    Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several “moltinism” mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval–larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval–pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH–deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis. PMID:22412378

  7. Ligand carrier protein genes expressed in larval chemosensory organs of Bombyx mori.

    PubMed

    Yoshizawa, Yasutaka; Sato, Ryoichi; Tsuchihara, Kazuko; Ozaki, Katsuhisa; Mita, Kazuei; Asaoka, Kiyoshi; Taniai, Kiyoko

    2011-08-01

    Expressed sequence tags (ESTs) of the maxillary galea of the silkworm were analyzed to identify proteins involved in food selection systems. From the 1251 redundant genes of the ESTs, we identified 7 odorant-binding protein-like genes (bmObpL), 6 takeout-like genes (bmToL), and 6 chemosensory protein genes (bmCsp). Quantitative RT-PCR analysis indicated that bmObpL1, bmObpL2, bmObpL3, bmObpL5, bmToL1, bmToL3, and bmorCsp15 were predominantly expressed in the larval oral appendages, such as the maxilla, labrum, labium and antenna. Immunocytochemical analysis indicated that the proteins of bmObpL1, bmObpL3, and bmToL1 were localized in the gustatory chemosensilla on the maxillary galea and olfactory sensilla in the antenna. The proteins encoded by bmObpL1 and bmObpL3 were detected in the gustatory chemosensilla of the epipharynx. However, bmObpL1 and bmToL1 were also detected in tactile hairs and in the epidermis of several chemosensory organs. The bmObpL2 protein was localized inside and in the epidermis around the chemosensilla, tactile hairs, and wide surface of the epipharynx. From these results, bmObpL3 is the most likely to have a dedicated role in chemoreception in the silkworm, Bombyx mori. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. Molecular cloning, characterization and expression analysis of ATG1 in the silkworm, Bombyx mori.

    PubMed

    Casati, Barbara; Terova, Genciana; Cattaneo, Anna Giulia; Rimoldi, Simona; Franzetti, Eleonora; de Eguileor, Magda; Tettamanti, Gianluca

    2012-12-15

    Atg1 is a Serine/Threonine protein kinase that plays a pivotal role in autophagy. A complete coding sequence of ATG1 is not available for the silkworm, Bombyx mori which is a good model for studying the autophagic process. In the present study we isolated two full-length cDNAs of 2175 (transcript variant A) and 2271 (transcript variant B) bases representing ATG1 in the silkworm. Phylogenetic analysis indicated that BmATG1 was closely related to orthologs of other insects. The encoded BmAtg1 proteins shared extensive homology with orthologs from yeast to mammals, showing high conservation at the N-terminal region where the catalytic domain and ATP- and Mg-binding sites are located. A de novo prediction of the three-dimensional structure for each protein is presented. We used real-time RT-PCR to quantify dynamic changes in mRNA copy number of BmATG1 in the midgut and fat body of fifth instar larvae undergoing starvation, as well as in other tissues of silkworm at the end of last larval instar. Our qPCR results revealed that BmATG1 expression levels at the end of larval life were comparable in the midgut, fat body and Malpighian tubules, while these were higher in the gonads; moreover, the mRNA copy number of ATG1 was very different among the anterior, middle and posterior silk glands. Real-time PCR analysis also showed that starvation significantly influenced BmATG1 mRNA copy number in the fat body of silkworm, inducing an upregulation 24h after food withdrawal, with only a slight effect in the midgut. Low expression levels of BmATG1 were observed in both tissues of control animals up to the second day of spinning phase. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. The Effect of Bovine Milk on the Growth of Bombyx mori

    PubMed Central

    Konala, Niharika; Abburi, Praveena; Bovilla, Venugopal Reddy; Mamillapalli, Anitha

    2013-01-01

    Bombyx mori L. (Lepidoptera: Bombycidae) is a well-studied Lepidopteran model system because of its morphology, life cycle, and economic importance. Many scientists have placed importance on enhancing the economic traits of B. mori because it's larvae, silkworms, are vital in the production of silk. In this study, the effect of bovine milk on B. mori growth was tested. Bovine milk contains several components that aid in healthy growth. The treatment was given to fifth instar B. mori larvae because the fifth instar period is when B. mori eats voraciously and shows maximum growth among all its larval stages. The larvae were treated with fresh mulberry, Morus L. (Rosales: Moraceae), leaves and mulberry leaves dipped in milk from the first day of the fifth instar. Treatments were given on alternate days, and the silkworms were weighed every day to determine whether milk had any role in enhancing the weight of the larvae. Cocoon weights were measured, as the weight indicates the approximate amount of silk that can be reeled. The results showed that larvae gained 82.5% more weight by the end of fifth instar larval when fed with mulberry leaves dipped in milk than when fed with fresh mulberry leaves without milk. The larvae fed with milk-treated leaves gained 310% weight from day 1 to day 7 of the fifth instar, while the larvae fed with fresh leaves gained 153% weight in the same timespan. In addition, cocoon weight increased by 8% when milk was added compared to when it was not. These results suggest that B. mori larvae can be fed mulberry leaves treated with bovine milk for better growth rate and increased silk production. PMID:24205942

  10. Transcriptional Profiling of Midgut Immunity Response and Degeneration in the Wandering Silkworm, Bombyx mori

    PubMed Central

    Xiao, Guohua; Yang, Bing; Zhang, Jie; Li, Xuquan; Guan, Jingmin; Shao, Qimiao; Beerntsen, Brenda T.; Zhang, Peng; Wang, Chengshu; Ling, Erjun

    2012-01-01

    Background Lepidoptera insects have a novel development process comprising several metamorphic stages during their life cycle compared with vertebrate animals. Unlike most Lepidoptera insects that live on nectar during the adult stage, the Bombyx mori silkworm adults do not eat anything and die after egg-laying. In addition, the midguts of Lepidoptera insects produce antimicrobial proteins during the wandering stage when the larval tissues undergo numerous changes. The exact mechanisms responsible for these phenomena remain unclear. Principal Findings We used the silkworm as a model and performed genome-wide transcriptional profiling of the midgut between the feeding stage and the wandering stage. Many genes concerned with metabolism, digestion, and ion and small molecule transportation were down-regulated during the wandering stage, indicating that the wandering stage midgut loses its normal functions. Microarray profiling, qRT-PCR and western blot proved the production of antimicrobial proteins (peptides) in the midgut during the wandering stage. Different genes of the immune deficiency (Imd) pathway were up-regulated during the wandering stage. However, some key genes belonging to the Toll pathway showed no change in their transcription levels. Unlike butterfly (Pachliopta aristolochiae), the midgut of silkworm moth has a layer of cells, indicating that the development of midgut since the wandering stage is not usual. Cell division in the midgut was observed only for a short time during the wandering stage. However, there was extensive cell apoptosis before pupation. The imbalance of cell division and apoptosis probably drives the continuous degeneration of the midgut in the silkworm since the wandering stage. Conclusions This study provided an insight into the mechanism of the degeneration of the silkworm midgut and the production of innate immunity-related proteins during the wandering stage. The imbalance of cell division and apoptosis induces irreversible

  11. Analysis of codon usage bias of mitochondrial genome in Bombyx mori and its relation to evolution.

    PubMed

    Wei, Lei; He, Jian; Jia, Xian; Qi, Qi; Liang, Zhisheng; Zheng, Hao; Ping, Yao; Liu, Shuyu; Sun, Jingchen

    2014-12-17

    Synonymous codon usage bias (SCUB) is an inevitable phenomenon in organismic taxa, generally referring to differences in the occurrence frequency of codons across different species or within the genome of the same species. SCUB happens in various degrees under pressure from nature selection, mutation bias and other factors in different ways. It also attaches great significance to gene expression and species evolution, however, a systematic investigation towards the codon usage in Bombyx mori (B. mori) has not been reported yet. Moreover, it is still indistinct about the reasons contributing to the bias or the relationship between the bias and the evolution of B. mori. The comparison of the codon usage pattern between the genomic DNA (gDNA) and the mitochondrial DNA (mtDNA) from B. mori suggests that mtDNA has a higher level of codon bias. Furthermore, the correspondence analysis suggests that natural selection, such as gene length, gene function and translational selection, dominates the codon preference of mtDNA, while the composition constraints for mutation bias only plays a minor role. Additionally, the clustering results of the silkworm superfamily suggest a lack of explicitness in the relationship between the codon usage of mitogenome and species evolution. Among the complicated influence factors leading to codon bias, natural selection is found to play a major role in shaping the high bias in the mtDNA of B. mori from our current data. Although the cluster analysis reveals that codon bias correlates little with the species evolution, furthermore, a detailed analysis of codon usage of mitogenome provides better insight into the evolutionary relationships in Lepidoptera. However, more new methods and data are needed to investigate the relationship between the mtDNA bias and evolution.

  12. Temperature effect on the recovery process in stretched Bombyx mori silk fibers

    NASA Astrophysics Data System (ADS)

    Aksakal, Baki

    2016-01-01

    The recovery process in stretched Bombyx mori silk fibers at different strain levels from 3% to 17% was investigated at room conditions during long period of time from 5 min to 20 days and more. How the temperature affects the recovery process in the silk fibers stretched at room conditions was examined at temperatures from 25 to 125 °C. The results of the recovery process at 25 °C revealed that although the recovery process from strain values higher than 3% strain continued slowly which caused quite high remaining deformation, a complete recovery from 3% strain was observed after 3 days. However, better recovery process was observed with increasing temperature which led to lower remaining deformations. For instance, a complete recovery from 6% strain was observed after 144 h and 3 h for the recovery process at 100 °C and 125 °C, respectively which indicates an important result that the deformations induced by stretching the silk fibers up to 6% strain are reversible and increasing temperature affects the velocity of this process significantly. The recovery process expressed in the strain (ε) and logarithm time coordinates showed a linear dependence for which a linear equation was proposed. Thus, this linear equation enables to estimate the required time for a complete recovery from different strain levels and remaining deformation at any stage of the recovery at different temperatures. The ATR-FTIR spectra of the stretched silk fibers during the recovery process revealed some changes in the absorbance ratios and shifts in the positions of the bands assigned to Cα-C, N-H stretching vibrations, and the Amide III mode. It was suggested that new formation of the hydrogen bonds between polypeptide chains especially in amorphous regions and the changes in the intra-sheet hydrogen bonds in β-sheet crystalline regions greatly contribute to the recovery process.

  13. Mandarin Chinese Dictionary: English-Chinese.

    ERIC Educational Resources Information Center

    Wang, Fred Fangyu

    This dictionary is a companion volume to the "Mandarin Chinese Dictionary (Chinese-English)" published in 1967 by Seton Hall University. The purpose of the dictionary is to help English-speaking students produce Chinese sentences in certain cultural situations by looking up the English expressions. Natural, spoken Chinese expressions within the…

  14. Mandarin Chinese Dictionary: English-Chinese.

    ERIC Educational Resources Information Center

    Wang, Fred Fangyu

    This dictionary is a companion volume to the "Mandarin Chinese Dictionary (Chinese-English)" published in 1967 by Seton Hall University. The purpose of the dictionary is to help English-speaking students produce Chinese sentences in certain cultural situations by looking up the English expressions. Natural, spoken Chinese expressions within the…

  15. Chinese Connections

    ERIC Educational Resources Information Center

    Skilling, William C.

    2012-01-01

    When L. Brooks Patterson, the executive of Oakland County, Michigan, publicly called for the county to become the first in America to teach Mandarin Chinese in every public school district, the Oxford Community Schools responded immediately. Over the past four years, the school district of 5,030 students in southeastern Michigan has elevated the…

  16. New insights into the catalytic mechanism of Bombyx mori prostaglandin E synthase gained from structure–function analysis

    SciTech Connect

    Yamamoto, Kohji; Suzuki, Mamoru; Higashiura, Akifumi; Aritake, Kosuke; Urade, Yoshihiro; Uodome, Nobuko; Hossain, MD. Tofazzal; Nakagawa, Atsushi

    2013-11-01

    Highlights: •Structure of Bombyx mori prostaglandin E synthase is determined. •Bound glutathione sulfonic acid is located at the glutathione-binding site. •Electron-sharing network is present in this protein. •This network includes Asn95, Asp96, and Arg98. •Site-directed mutagenesis reveals that the residues contribute to the catalytic activity. -- Abstract: Prostaglandin E synthase (PGES) catalyzes the isomerization of PGH{sub 2} to PGE{sub 2}. We previously reported the identification and structural characterization of Bombyx mori PGES (bmPGES), which belongs to Sigma-class glutathione transferase. Here, we extend these studies by determining the structure of bmPGES in complex with glutathione sulfonic acid (GTS) at a resolution of 1.37 Å using X-ray crystallography. GTS localized to the glutathione-binding site. We found that electron-sharing network of bmPGES includes Asn95, Asp96, and Arg98. Site-directed mutagenesis of these residues to create mutant forms of bmPGES mutants indicate that they contribute to catalytic activity. These results are, to our knowledge, the first to reveal the presence of an electron-sharing network in bmPGES.

  17. Cloning and characterization of carboxyl terminus of heat shock cognate 70-interacting protein gene from the silkworm, Bombyx mori.

    PubMed

    Ohsawa, Takeshi; Fujimoto, Shota; Tsunakawa, Akane; Shibano, Yuka; Kawasaki, Hideki; Iwanaga, Masashi

    2016-11-01

    Carboxyl terminus of heat shock cognate 70-interacting protein (CHIP) is an evolutionarily conserved E3 ubiquitin ligase across different eukaryotic species and is known to play a key role in protein quality control. CHIP has two distinct functional domains, an N-terminal tetratricopeptide repeat (TPR) and a C-terminal U-box domain, which are required for the ubiquitination of numerous labile client proteins that are chaperoned by heat shock proteins (HSPs) and heat shock cognate proteins (HSCs). During our screen for CHIP-like proteins in the Bombyx mori databases, we found a novel silkworm gene, Bombyx mori CHIP. Phylogenetic analysis showed that BmCHIP belongs to Lepidopteran lineages. Quantitative reverse transcription-PCR analysis indicated that BmCHIP was relatively highly expressed in the gonad and fat body. A pull-down experiment and auto-ubiquitination assay showed that BmCHIP interacted with BmHSC70 and had E3 ligase activity. Additionally, immunohistochemical analysis revealed that BmCHIP was partially co-localized with ubiquitin in BmN4 cells. These data support that BmCHIP plays an important role in the ubiquitin proteasome system as an E3 ubiquitin ligase in B. mori. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. A Review of the Implications of Heterozygosity and Inbreeding on Germplasm Biodiversity and Its Conservation in the Silkworm, Bombyx mori

    PubMed Central

    Jingade, A.H.; Vijayan, K.; Somasundaram, P.; Srivasababu, G.K.; Kamble, C.K.

    2011-01-01

    Silkworm genebanks assume paramount importance as the reservoirs of biodiversity and source of alleles that can be easily retrieved for genetic enhancement of popular breeds. More than 4000 Bombyx mori L (Lepidoptera: Bombycidae) strains are currently available and these strains are maintained through continuous sibling mating. This repeated sibling mating makes the populations of each strain more homozygous, but leads to loss of unique and valuable genes through the process of inbreeding depression. Hence, it is essential to maintain a minimal degree of heterozygosity within the population of each silkworm strain, especially in the traditional geographic strains, to avoid such loss. As a result, accurate estimation of genetic diversity is becoming more important in silkworm genetic resources conservation. Application of molecular markers help estimate genetic diversity much more accurately than that of morphological traits. Since a minimal amount of heterozygosity in each silkworm strain is essential for better conservation by avoiding inbreeding depression, this article overviews both theoretical and practical importance of heterozygosity together with impacts of inbreeding depression and the merits and demerits of neutral molecular markers for measurements of both heterozygosity and inbreeding depression in the silkworm Bombyx mori. PMID:21521139

  19. A review of the implications of heterozygosity and inbreeding on germplasm biodiversity and its conservation in the silkworm, Bombyx mori.

    PubMed

    Jingade, A H; Vijayan, K; Somasundaram, P; Srivasababu, G K; Kamble, C K

    2011-01-01

    Abstract Silkworm genebanks assume paramount importance as the reservoirs of biodiversity and source of alleles that can be easily retrieved for genetic enhancement of popular breeds. More than 4000 Bombyx mori L (Lepidoptera: Bombycidae) strains are currently available and these strains are maintained through continuous sibling mating. This repeated sibling mating makes the populations of each strain more homozygous, but leads to loss of unique and valuable genes through the process of inbreeding depression. Hence, it is essential to maintain a minimal degree of heterozygosity within the population of each silkworm strain, especially in the traditional geographic strains, to avoid such loss. As a result, accurate estimation of genetic diversity is becoming more important in silkworm genetic resources conservation. Application of molecular markers help estimate genetic diversity much more accurately than that of morphological traits. Since a minimal amount of heterozygosity in each silkworm strain is essential for better conservation by avoiding inbreeding depression, this article overviews both theoretical and practical importance of heterozygosity together with impacts of inbreeding depression and the merits and demerits of neutral molecular markers for measurements of both heterozygosity and inbreeding depression in the silkworm Bombyx mori.

  20. Mapping of sex-linked genes onto the genome sequence using various aberrations of the Z chromosome in Bombyx mori.

    PubMed

    Fujii, Tsuguru; Abe, Hiroaki; Katsuma, Susumu; Mita, Kazuei; Shimada, Toru

    2008-12-01

    Many strains of Bombyx mori carry chromosomal aberrations, and they are useful resources for integration between phenotypes and genomic sequences. We compared the molecular structures of three kinds of Z chromosomes, i.e., two strains with chromosome deletions and one strain with translocation involving the Z chromosome. Using polymerase chain reaction markers, we showed that: (1) the Z(1) chromosome lacks more than 6Mb, including the proximal end; (2) the Z(Vg) chromosome lacks 1.5Mb in the interstitial portion; and (3) the +(od)p(Sa)+(p)W carries a 0.6-Mb Z-derived fragment surrounding the +(od) gene. The breakpoint junctions of these deletions and a translocation were precisely determined. Through deletion mapping, we narrowed down the regions where distinct oily (od), vestigial (Vg), and muscle dystrophy (Md) are located and identified a candidate gene for od. A retroposon-mediated deletion in BmBLOS2--the Bombyx gene homologous to human "biogenesis of lysosome-related organelles complex-1, subunit 2''--was detected in the od mutant. Although the genes responsible for Vg and Md were not definitively identified, we propose the candidate genes on the basis of their locations and phenotypes.

  1. Comparisons of contact chemoreception and food acceptance by larvae of polyphagous Helicoverpa armigera and oligophagous Bombyx mori.

    PubMed

    Zhang, Hui-Jie; Faucher, Cécile P; Anderson, Alisha; Berna, Amalia Z; Trowell, Stephen; Chen, Quan-Mei; Xia, Qing-You; Chyb, Sylwester

    2013-08-01

    We compared food choice and the initial response to deterrent treated diet between fifth instars of Helicoverpa armigera, a polyphagous generalist pest, and Bombyx mori, an oligophagous specialist beneficial. Bombyx mori was more behaviorally sensitive to salicin than to caffeine. The relative sensitivities were reversed for H. armigera, which was tolerant to the highest levels of salicin found in natural sources but sensitive to caffeine. A single gustatory receptor neuron (GRN) in the medial styloconic sensillum of B. mori was highly sensitive to salicin and caffeine. The styloconic sensilla of H. armigera did not respond consistently to either of the bitter compounds. Phagostimulants also were tested. Myo-inositol and sucrose were detected specifically by two GRNs located in B. mori lateral styloconic sensillum, whereas, in H. armigera, sucrose was sensed by a GRN in the lateral sensillum, and myo-inositol by a GRN in the medial sensillum. Myo-inositol responsiveness in both species occurred at or below 10(-3) mM, which is far below the naturally occurring concentration of 1 mM in plants. Larval responses to specific plant secondary compounds appear to have complex determinants that may include host range, metabolic capacity, and gustatory repertoire.

  2. Transcriptional regulation of the insulin signaling pathway genes by starvation and 20-hydroxyecdysone in the Bombyx fat body.

    PubMed

    Liu, Yan; Zhou, Shun; Ma, Li; Tian, Ling; Wang, Sheng; Sheng, Zhentao; Jiang, Rong-Jing; Bendena, William G; Li, Sheng

    2010-10-01

    Genetic studies in the fruitfly, Drosophila melanogaster, have uncovered a conserved insulin/insulin growth factor signaling (IIS) pathway that regulates nutrition-dependent growth rates of insects. From the silkworm, Bombyx mori, we have identified and characterized several key genes involved in the IIS pathway, including InR, IRS, PI3K110, PI3K60, PTEN, PDK, and Akt. Tissue distribution analysis showed that most of these genes were highly expressed in the fat body implying that the IIS pathway is functionally important within insect adipose tissue. Developmental profile studies revealed that the expression levels of InR, IRS, PI3K110, and PDK were elevated in the fat body during molting and pupation, periods when animals ceased feeding and hemolymph levels of 20-hydroxyecdysone (20E) were high. Starvation rapidly up-regulated the mRNA levels of these same genes in the fat body, while 20E slowly induced their transcription. We conclude that 20E slowly reduces food consumption and then indirectly induces a state of starvation resulting in the elevation of the mRNA levels of InR, IRS, PI3K110, and PDK in the Bombyx fat body during molting and pupation. Copyright 2010 Elsevier Ltd. All rights reserved.

  3. ERK/MAPK regulates ecdysteroid and sorbitol metabolism for embryonic diapause termination in the silkworm, Bombyx mori.

    PubMed

    Fujiwara, Yoshihiro; Tanaka, Yoshiaki; Iwata, Ken-ichi; Rubio, Raquel O; Yaginuma, Toshinobu; Yamashita, Okitsugu; Shiomi, Kunihiro

    2006-06-01

    The eggs of the silkworm Bombyx mori undergo a state of suspended overt development and diminished metabolism called "diapause" to escape adverse environmental conditions. Termination of Bombyx embryonic diapause requires 2-3 months of low temperature (5 degrees C), but the molecular mechanisms underlying diapause termination are unknown. Diapause termination requires a decrease in the sorbitol concentration, which arrests embryonic development, and the secretion factors from yolk cells that promote embryonic development. In the present study, we report that 20-hydroxyecdysone promoted the development of denuded embryos and that ecdysteroid-phosphate phosphatase (EPPase), which is a key enzyme for active ecdysteroid production, was induced by incubation of diapausing eggs at 5 degrees C. In dechorionated egg cultures, extracellular signal-regulated kinase (ERK), which is activated by incubating diapausing eggs at 5 degrees C, regulated sorbitol-glycogen conversion, ecdysteroid secretion via gene transcription of key enzymes, sorbitol dehydrogenase-2, and EPPase, suggesting that ERK has a key role in diapause termination.

  4. Chinese Geography through Chinese Cuisine

    ERIC Educational Resources Information Center

    Lipman, Jonathan

    2010-01-01

    China has the world's largest population, now over 1.3 billion, but its land area (much of it high mountains or desert) is about the same as that of the United States, which has less than one-fourth as many people. So Chinese farmers have learned to use every inch of their fertile land intensively. Pressure on the land has required extremely…

  5. Chinese Geography through Chinese Cuisine

    ERIC Educational Resources Information Center

    Lipman, Jonathan

    2010-01-01

    China has the world's largest population, now over 1.3 billion, but its land area (much of it high mountains or desert) is about the same as that of the United States, which has less than one-fourth as many people. So Chinese farmers have learned to use every inch of their fertile land intensively. Pressure on the land has required extremely…

  6. Genome Sequence of a Novel Iflavirus from mRNA Sequencing of the Pupa of Bombyx mori Inoculated with Cordyceps militaris

    PubMed Central

    Suzuki, Tomohiro; Takeshima, Yoshino; Mikamoto, Toshiyuki; Saeki, Jun-David; Kato, Tatsuya; Park, Enoch Y.

    2015-01-01

    We discovered a novel iflavirus from the transcriptome of the Bombyx mori pupa inoculated with the insect-pathogenic fungus Cordyceps militaris. The assembled iflavirus genome has 10,119 nucleotides, with a 3′-polyadenylated tail, and it encodes a polyprotein composed of 3,004 amino acids. PMID:26383664

  7. Identification of specific sites in the third intracellular loop and carboxyl terminus of the Bombyx mori PBAN receptor crucial for ligand-induced internalization

    USDA-ARS?s Scientific Manuscript database

    Sex pheromone production in most moths is mediated by the pheromone biosynthesis activating neuropeptide receptor (PBANR). Similar to other rhodopsin-like G protein-coupled receptors, the silkmoth Bombyx mori PBANR (BmPBANR) undergoes agonist-induced internalization. Despite interest in developing...

  8. Genome Sequence of a Novel Iflavirus from mRNA Sequencing of the Pupa of Bombyx mori Inoculated with Cordyceps militaris.

    PubMed

    Suzuki, Tomohiro; Takeshima, Yoshino; Mikamoto, Toshiyuki; Saeki, Jun-David; Kato, Tatsuya; Park, Enoch Y; Kawagishi, Hirokazu; Dohra, Hideo

    2015-09-17

    We discovered a novel iflavirus from the transcriptome of the Bombyx mori pupa inoculated with the insect-pathogenic fungus Cordyceps militaris. The assembled iflavirus genome has 10,119 nucleotides, with a 3'-polyadenylated tail, and it encodes a polyprotein composed of 3,004 amino acids.

  9. Discerning Silk Produced by Bombyx mori from Those Produced by Wild Species Using an Enzyme-Linked Immunosorbent Assay Combined with Conventional Methods.

    PubMed

    You, Qiushi; Li, Qingqing; Zheng, Hailing; Hu, Zhiwen; Zhou, Yang; Wang, Bing

    2017-09-06

    Recently, much interest has been paid to the separation of silk produced by Bombyx mori from silk produced by other species and tracing the beginnings of silk cultivation from wild silk exploitation. In this paper, significant differences between silks from Bombyx mori and other species were found by microscopy and spectroscopy, such as morphology, secondary structure, and amino acid composition. For further accurate identification, a diagnostic antibody was designed by comparing the peptide sequences of silks produced by Bombyx mori and other species. The results of the noncompetitive indirect enzyme-linked immunosorbent assay (ELISA) indicated that the antibody that showed good sensitivity and high specificity can definitely discern silk produced by Bombyx mori from silk produced by wild species. Thus, the antibody-based immunoassay has the potential to be a powerful tool for tracing the beginnings of silk cultivation. In addition, combining the sensitive, specific, and convenient ELISA technology with other conventional methods can provide more in-depth and accurate information for species identification.

  10. Partial deletions of the W chromosome due to reciprocal translocation in the silkworm Bombyx mori.

    PubMed

    Abe, H; Seki, M; Ohbayashi, F; Tanaka, N; Yamashita, J; Fujii, T; Yokoyama, T; Takahashi, M; Banno, Y; Sahara, K; Yoshido, A; Ihara, J; Yasukochi, Y; Mita, K; Ajimura, M; Suzuki, M G; Oshiki, T; Shimada, T

    2005-08-01

    In the silkworm, Bombyx mori (female, ZW; male, ZZ), femaleness is determined by the presence of a single W chromosome, irrespective of the number of autosomes or Z chromosomes. The W chromosome is devoid of functional genes, except the putative female-determining gene (Fem). However, there are strains in which chromosomal fragments containing autosomal markers have been translocated on to W. In this study, we analysed the W chromosomal regions of the Zebra-W strain (T(W;3)Ze chromosome) and the Black-egg-W strain (T(W;10)+(w-2) chromosome) at the molecular level. Initially, we undertook a project to identify W-specific RAPD markers, in addition to the three already established W-specific RAPD markers (W-Kabuki, W-Samurai and W-Kamikaze). Following the screening of 3648 arbitrary 10-mer primers, we obtained nine W-specific RAPD marker sequences (W-Bonsai, W-Mikan, W-Musashi, W-Rikishi, W-Sakura, W-Sasuke, W-Yukemuri-L, W-Yukemuri-S and BMC1-Kabuki), almost all of which contained the border regions of retrotransposons, namely portions of nested retrotransposons. We confirmed the presence of eleven out of twelve W-specific RAPD markers in the normal W chromosomes of twenty-five silkworm strains maintained in Japan. These results indicate that the W chromosomes of the strains in Japan are almost identical in type. The Zebra-W strain (T(W;3)Ze chromosome) lacked the W-Samurai and W-Mikan RAPD markers and the Black-egg-W strain (T(W;10)+(w-2) chromosome) lacked the W-Mikan RAPD marker. These results strongly indicate that the regions containing the W-Samurai and W-Mikan RAPD markers or the W-Mikan RAPD marker were deleted in the T(W;3)Ze and T(W;10)+(w-2) chromosomes, respectively, due to reciprocal translocation between the W chromosome and the autosome. This deletion apparently does not affect the expression of Fem; therefore, this deleted region of the W chromosome does not contain the putative Fem gene.

  11. 20-hydroxyecdysone and juvenile hormone analog prevent precocious metamorphosis in recessive trimolter mutants of Bombyx mori.

    PubMed

    Wang, Hua-Bing; Ali, Saheb Md; Moriyama, Minoru; Iwanaga, Masashi; Kawasaki, Hideki

    2012-02-01

    The trimolter mutants of Bombyx mori have four instead of five larval instars of normal tetramolters. Here, we show that the tetramolter was induced in the recessive trimolter European No.7 mutant (rt-E7) by application of either the juvenile hormone analog (JHA) or 20-hydroxyecdysone (20E). However, treatments with JHA or 20E did not change the number of larval instars of the dominant trimolter Si Chuan mutant (DT-SC). Krüppel-homolog 1 (Kr-h1) is an early JH-response gene that mediates the anti-metamorphic action of JH. In the wing disc of tetramolter B. mori, Kr-h1 RNAs decreased shortly after ecdysis to the fifth instar, while pupal specifier gene, Broad Complex Z1 (BR-Z1) RNAs slightly increased and coincided with the onset of metamorphic competence of wing discs. Analysis of the developmental profile of Kr-h1 in the wing disc of rt-E7 showed that its transcript slightly increased from 12 to 24 h and gradually decreased between 24 and 72 h in the fourth (last) larval instar, while Kr-h1 mRNA decreased rapidly between 12 and 72 h in DT-SC. In addition, the expression of BR-Z1 in DT-SC during the early fourth (last) larval instar is relatively higher than that in rt-E7. These results indicated that the occurrence of pupal commitment of the wing disc in DT-SC was much earlier than that in rt-E7. In the early fourth larval instar of rt-E7, feeding on 20E or treatments with exogenous JHA caused up-regulation of Kr-h1, suppressed premature induction of BR-Z1, and then induced an additional larval instar. By contrast, in DT-SC mutant, since pupal commitment immediately occurred after third ecdysis, precocious metamorphosis was not successfully rescued. The results suggest that Kr-h1 and BR-Z1 involved in the prevention of precocious metamorphosis in recessive trimolter mutants by application of 20E and JHA. The result indicated that Kr-h1 and BR-Z1 expression reflected larval-pupal transition of the recessive trimolter of B. mori. Copyright © 2011 Elsevier Ltd

  12. Systematic Identification and Characterization of Long Non-Coding RNAs in the Silkworm, Bombyx mori

    PubMed Central

    Wu, Yuqian; Cheng, Tingcai; Liu, Chun; Liu, Duolian; Zhang, Quan; Long, Renwen; Zhao, Ping; Xia, Qingyou

    2016-01-01

    Long noncoding RNAs (lncRNAs) are emerging as important regulators in various biological processes. However, to date, no systematic characterization of lncRNAs has been reported in the silkworm Bombyx mori. In the present study, we generated eighteen RNA-seq datasets with relatively high depth. Using an in-house designed lncRNA identification pipeline, 11,810 lncRNAs were identified for 5,556 loci. Among these lncRNAs, 474 transcripts were intronic lncRNAs (ilncRNAs), 6,250 transcripts were intergenic lncRNAs (lincRNAs), and 5,086 were natural antisense lncRNAs (lncNATs). Compared with protein-coding mRNAs, silkworm lncRNAs are shorter in terms of full length but longer in terms of exon and intron length. In addition, lncRNAs exhibit a lower level of sequence conservation, more repeat sequences overlapped and higher tissue specificity than protein-coding mRNAs in the silkworm. We found that 69 lncRNA transcripts from 33 gene loci may function as miRNA precursors, and 104 lncRNA transcripts from 72 gene loci may act as competing endogenous RNAs (ceRNAs). In total, 49.47% of all gene loci (2,749/5,556) for which lncRNAs were identified showed sex-biased expression. Co-expression network analysis resulted in 19 modules, 12 of which revealed relatively high tissue specificity. The highlighted darkgoldenrod module was specifically associated with middle and posterior silk glands, and the hub lncRNAs within this module were co-expressed with proteins involved in translation, translocation, and secretory processes, suggesting that these hub lncRNAs may function as regulators of the biosynthesis, translocation, and secretion of silk proteins. This study presents the first comprehensive genome-wide analysis of silkworm lncRNAs and provides an invaluable resource for genetic, evolutionary, and genomic studies of B. mori. PMID:26771876

  13. Systematic Identification and Characterization of Long Non-Coding RNAs in the Silkworm, Bombyx mori.

    PubMed

    Wu, Yuqian; Cheng, Tingcai; Liu, Chun; Liu, Duolian; Zhang, Quan; Long, Renwen; Zhao, Ping; Xia, Qingyou

    2016-01-01

    Long noncoding RNAs (lncRNAs) are emerging as important regulators in various biological processes. However, to date, no systematic characterization of lncRNAs has been reported in the silkworm Bombyx mori. In the present study, we generated eighteen RNA-seq datasets with relatively high depth. Using an in-house designed lncRNA identification pipeline, 11,810 lncRNAs were identified for 5,556 loci. Among these lncRNAs, 474 transcripts were intronic lncRNAs (ilncRNAs), 6,250 transcripts were intergenic lncRNAs (lincRNAs), and 5,086 were natural antisense lncRNAs (lncNATs). Compared with protein-coding mRNAs, silkworm lncRNAs are shorter in terms of full length but longer in terms of exon and intron length. In addition, lncRNAs exhibit a lower level of sequence conservation, more repeat sequences overlapped and higher tissue specificity than protein-coding mRNAs in the silkworm. We found that 69 lncRNA transcripts from 33 gene loci may function as miRNA precursors, and 104 lncRNA transcripts from 72 gene loci may act as competing endogenous RNAs (ceRNAs). In total, 49.47% of all gene loci (2,749/5,556) for which lncRNAs were identified showed sex-biased expression. Co-expression network analysis resulted in 19 modules, 12 of which revealed relatively high tissue specificity. The highlighted darkgoldenrod module was specifically associated with middle and posterior silk glands, and the hub lncRNAs within this module were co-expressed with proteins involved in translation, translocation, and secretory processes, suggesting that these hub lncRNAs may function as regulators of the biosynthesis, translocation, and secretion of silk proteins. This study presents the first comprehensive genome-wide analysis of silkworm lncRNAs and provides an invaluable resource for genetic, evolutionary, and genomic studies of B. mori.

  14. Expression map of a complete set of gustatory receptor genes in chemosensory organs of Bombyx mori.

    PubMed

    Guo, Huizhen; Cheng, Tingcai; Chen, Zhiwei; Jiang, Liang; Guo, Youbing; Liu, Jianqiu; Li, Shenglong; Taniai, Kiyoko; Asaoka, Kiyoshi; Kadono-Okuda, Keiko; Arunkumar, Kallare P; Wu, Jiaqi; Kishino, Hirohisa; Zhang, Huijie; Seth, Rakesh K; Gopinathan, Karumathil P; Montagné, Nicolas; Jacquin-Joly, Emmanuelle; Goldsmith, Marian R; Xia, Qingyou; Mita, Kazuei

    2017-03-01

    Most lepidopteran species are herbivores, and interaction with host plants affects their gene expression and behavior as well as their genome evolution. Gustatory receptors (Grs) are expected to mediate host plant selection, feeding, oviposition and courtship behavior. However, due to their high diversity, sequence divergence and extremely low level of expression it has been difficult to identify precisely a complete set of Grs in Lepidoptera. By manual annotation and BAC sequencing, we improved annotation of 43 gene sequences compared with previously reported Grs in the most studied lepidopteran model, the silkworm, Bombyx mori, and identified 7 new tandem copies of BmGr30 on chromosome 7, bringing the total number of BmGrs to 76. Among these, we mapped 68 genes to chromosomes in a newly constructed chromosome distribution map and 8 genes to scaffolds; we also found new evidence for large clusters of BmGrs, especially from the bitter receptor family. RNA-seq analysis of diverse BmGr expression patterns in chemosensory organs of larvae and adults enabled us to draw a precise organ specific map of BmGr expression. Interestingly, most of the clustered genes were expressed in the same tissues and more than half of the genes were expressed in larval maxillae, larval thoracic legs and adult legs. For example, BmGr63 showed high expression levels in all organs in both larval and adult stages. By contrast, some genes showed expression limited to specific developmental stages or organs and tissues. BmGr19 was highly expressed in larval chemosensory organs (especially antennae and thoracic legs), the single exon genes BmGr53 and BmGr67 were expressed exclusively in larval tissues, the BmGr27-BmGr31 gene cluster on chr7 displayed a high expression level limited to adult legs and the candidate CO2 receptor BmGr2 was highly expressed in adult antennae, where few other Grs were expressed. Transcriptional analysis of the Grs in B. mori provides a valuable new reference for

  15. Regeneration of Bombyx mori silk nanofibers and nanocomposite fibrils by the electrospinning process

    NASA Astrophysics Data System (ADS)

    Ayutsede, Jonathan Eyitouyo

    In recent years, there has been significant interest in the utilization of natural materials for novel nanoproducts such as tissue engineered scaffolds. Silkworm silk fibers represent one of the strongest natural fibers known. Silkworm silk, a protein-based natural biopolymer, has received renewed interest in recent years due to its unique properties (strength, toughness) and potential applications such as smart textiles, protective clothing and tissue engineering. The traditional 10--20 mum diameter, triangular-shaped Bombyx mori fibers have remained unchanged over the years. However, in our study, we examine the scientific implication and potential applications of reducing the diameter to the nanoscale, changing the triangular shape of the fiber and adding nanofillers in the form of single wall carbon nanotubes (SWNT) by the electrospinning process. The electrospinning process preserves the natural conformation of the silk (random and beta-sheet). The feasibility of changing the properties of the electrospun nanofibers by post processing treatments (annealing and chemical treatment) was investigated. B. mori silk fibroin solution (formic acid) was successfully electrospun to produce uniform nanofibers (as small as 12 nm). Response Surface Methodology (RSM) was applied for the first time to experimental results of electrospinning, to develop a processing window that can reproduce regenerated silk nanofibers of a predictable size (d < 100nm). SWNT-silk multifunctional nanocomposite fibers were fabricated for the first time with anticipated properties (mechanical, thermal and electrically conductive) that may have scientific applications (nerve regeneration, stimulation of cell-scaffold interaction). In order to realize these applications, the following areas need to be addressed: a systematic investigation of the dispersion of the nanotubes in the silk matrix, a determination of new methodologies for characterizing the nanofiber properties and establishing the

  16. Identification and characterization of two putative microRNAs encoded by Bombyx mori cypovirus.

    PubMed

    Pan, Zhong-Hua; Wu, Ping; Gao, Kun; Hou, Cheng-Xiang; Qin, Guang-Xing; Geng, Tao; Guo, Xi-Jie

    2017-04-02

    Viral microRNAs (miRNAs) have been demonstrated to play important roles in virus-host interactions. Some RNA virus-encoded miRNAs have been reported to promote viral replication and may be used as potential drug targets. Bombyx mori cypovirus (BmCPV), an important pathogen of silkworm, is a double-stranded RNA virus frequently causing serious damages in sericulture. Research on miRNA encoded by BmCPV may be useful to elucidate the BmCPV-host interaction and to develop new anti-viral methods. In our previous study, small RNA libraries of the midgut of BmCPV-infected silkworm have been generated by deep sequencing and several BmCPV-encoded putative miRNAs were predicted. In this study, two putative miRNAs encoded by BmCPV were identified and then validated by stem-loop qRT-PCR and northern blot. They are BmCPV-miR-3 encoded by the third genomic RNA segment of BmCPV (478-497bp) and BmCPV-miR-5 encoded by the fifth genomic RNA segment (2481-2500bp), both are 20bp and encoded by ORF regions. miRNA expression could be detected as early as 5h after BmCPV infection, and the expression level of BmCPV-miR-3 is much higher than that of BmCPV-miR-5 in the course of infection. Three potential target genes were predicted in the host genome, two for BmCPV-miR-3 and one for BmCPV-miR-5, but just one in the virus genome for BmCPV-miR-3 only, with the binding sites all in coding regions. Dual luciferase assay and qRT-PCR indicated that BmCPV-miR-3 could down-regulate the expression of both its two target genes, but no regulatory effect by BmCPV-miR-5 on its target gene was detected. In contrast, BmCPV-miR-3 could up-regulate the viral target. This is the first report that an insect double stranded RNA virus may generate miRNAs and the results obtained will benefit the future study of the functions of BmCPV-encoded miRNAs on viral replication and virus-host interaction. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Transcriptome Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection

    PubMed Central

    Kolliopoulou, Anna; Van Nieuwerburgh, Filip; Stravopodis, Dimitrios J.; Deforce, Dieter; Swevers, Luc; Smagghe, Guy

    2015-01-01

    Many insects can be persistently infected with viruses but do not show any obvious adverse effects with respect to physiology, development or reproduction. Here, Bombyx mori strain Daizo, persistently infected with cytoplasmic polyhedrosis virus (BmCPV), was used to study the host’s transcriptional response after pathogenic infection with the same virus in midgut tissue of larvae persistently and pathogenically infected as 2nd and 4th instars. Next generation sequencing revealed that from 13,769 expressed genes, 167 were upregulated and 141 downregulated in both larval instars following pathogenic infection. Several genes that could possibly be involved in B. mori immune response against BmCPV or that may be induced by the virus in order to increase infectivity were identified, whereas classification of differentially expressed transcripts (confirmed by qRT-PCR) resulted in gene categories related to physical barriers, immune responses, proteolytic / metabolic enzymes, heat-shock proteins, hormonal signaling and uncharacterized proteins. Comparison of our data with the available literature (pathogenic infection of persistently vs. non-persistently infected larvae) unveiled various similarities of response in both cases, which suggests that pre-existing persistent infection does not affect in a major way the transcriptome response against pathogenic infection. To investigate the possible host’s RNAi response against BmCPV challenge, the differential expression of RNAi-related genes and the accumulation of viral small RNAs (vsRNAs) were studied. During pathogenic infection, siRNA-like traces like the 2-fold up-regulation of the core RNAi genes Ago-2 and Dcr-2 as well as a peak of 20 nt small RNAs were observed. Interestingly, vsRNAs of the same size were detected at lower rates in persistently infected larvae. Collectively, our data provide an initial assessment of the relative significance of persistent infection of silkworm larvae on the host response following

  18. Molecular basis of female-specific odorant responses in Bombyx mori.

    PubMed

    Anderson, Alisha R; Wanner, Kevin W; Trowell, Stephen C; Warr, Coral G; Jaquin-Joly, Emmanuelle; Zagatti, Pierre; Robertson, Hugh; Newcomb, Richard D

    2009-03-01

    Males and females of many moth species exhibit important differences in sexual behaviours. Much research in this field has focused on the male-specific behaviour, electrophysiology and molecular biology of sex pheromone reception. Female-specific behaviours have been less well studied although, like male-specific behaviours, they could provide opportunities for intervention and management of lepidopteran pests. Previously, we identified genes encoding putative odorant receptors (ORs) from the genome of the silkworm, Bombyx mori, some of which have higher levels of steady-state transcript in the antennae of adult females compared with males. We have identified the full-length cDNA sequences of some of these ORs and described a novel OR that is part of a female-biased clade. Using expression in Sf9 cells and a calcium-imaging assay, we tested a range of compounds for their ability to activate the most highly female-biased ORs, BmOR19, BmOR30, BmOR45 and BmOR47. BmOR19 responds to linalool, while BmOR45 and BmOR47 respond to benzoic acid > 2-phenylethanol > benzaldehyde. No activating ligands were found for BmOR30. RNA in situ hybridisation experiments reveal that BmOR19 is expressed in female olfactory sensory neurons that are co-located in the same sensilla as a second ORN expressing BmOR45 and/or BmOR47. Taken together the activity and expression of these receptors is likely explanatory of the observed electrophysiology of long sensilla trichoidea of female B. mori, previously shown to each contain one terpene (BmOR19) and one benzoic acid (BmOR45, BmOR47) sensory neuron. Plant volatiles such as linalool, benzoic acid, 2-phenylethanol and benzaldehyde are oviposition cues for females of some moths. These compounds have also been found in male-produced pheromone blends extracted from the hair pencils of many noctuid species. Hair pencil structures have not previously been reported for B. mori, but we have found hair pencil-like structures in adult male B. mori that

  19. Expression of antimicrobial peptide genes in Bombyx mori gut modulated by oral bacterial infection and development.

    PubMed

    Wu, Shan; Zhang, Xiaofeng; He, Yongqiang; Shuai, Jiangbing; Chen, Xiaomei; Ling, Erjun

    2010-11-01

    Although Bombyx mori systematic immunity is extensively studied, little is known about the silkworm's intestine-specific responses to bacterial infection. Antimicrobial peptides (AMPs) gene expression analysis of B. mori intestinal tissue to oral infection with the Gram-positive (Staphylococcus aureus) and -negative (Escherichia coli) bacteria revealed that there is specificity in the interaction between host immune responses and parasite types. Neither Att1 nor Leb could be stimulated by S. aureus and E. coli. However, CecA1, Glo1, Glo2, Glo3, Glo4 and Lys, could only be trigged by S. aureus. On the contrary, E. coli stimulation caused the decrease in the expression of CecA1, Glo3 and Glo4 in some time points. Interestingly, there is regional specificity in the silkworm local gut immunity. During the immune response, the increase in Def, Hem and LLP3 was only detected in the foregut and midgut. For CecB1, CecD, LLP2 and Mor, after orally administered with E. coli, the up-regulation was only limited in the midgut and hindgut. CecE was the only AMP that positively responses to the both bacteria in all the testing situations. With development, the expression levels of the AMPs were also changed dramatically. That is, at spinning and prepupa stages, a large increase in the expression of CecA1, CecB1, CecD, CecE, Glo1, Glo2, Glo3, Glo4, Leb, Def, Hem, Mor and Lys was detected in the gut. Unexpectedly, in addition to the IMD pathway genes, the Toll and JAK/STAT pathway genes in the silkworm gut can also be activated by microbial oral infection. But in the developmental course, corresponding to the increase in expression of AMPs at spinning and prepupa stages, only the Toll pathway genes in the gut exhibit the similar increasing trend. Our results imply that the immune responses in the silkworm gut are synergistically regulated by the Toll, JAK/STAT and IMD pathways. However, as the time for approaching pupation, the Toll pathway may play a role in the AMPs expression.

  20. Chinese Ambition

    DTIC Science & Technology

    2000-04-01

    revolution training for example Chiang Kai- shek a future Chinese leader. Sun never fully reunified China nor gained control of northern provinces in his...lifetime but one of his lieutenants, Chiang Kai- shek (or Jiang Jieshi), did eventually gain control of all China at least nominally by 1928. Under the...population as the potential source for rebellion and struggled with the nationalist government of Chiang Kai- shek as early as 1921 to gain control of China

  1. iTRAQ-based quantitative proteomic analysis of midgut in silkworm infected with Bombyx mori cytoplasmic polyhedrosis virus.

    PubMed

    Gao, Kun; Deng, Xiang-Yuan; Shang, Meng-Ke; Qin, Guang-Xing; Hou, Cheng-Xiang; Guo, Xi-Jie

    2017-01-30

    Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects the epithelial cells in the midgut of silkworm and causes them to death, which negatively affects the sericulture industry. In order to determine the midgut response at the protein levels to the virus infection, differential proteomes of the silkworm midgut responsive to BmCPV infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). 193, 408, 189 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the midgut of BmCPV-infected and control larvae at 24, 48, 72h post infection (hpi) respectively. KEGG enrichment analysis showed that Oxidative phosphorylation, amyotrophic lateral sclerosis, Toll-like receptor signaling pathway, steroid hormone biosynthesis were the significant pathways (Q value≤0.05) both at 24 and 48hpi. qRT-PCR was used to further verify gene transcription of 30 DEPs from iTRAQ, showing that the regulations of 24 genes at the transcript level were consistent with those at the proteomic level. Moreover, the cluster analysis of the three time groups showed that there were seven co-regulated DEPs including BGIBMGA002620-PA, which was a putative p62/sequestosome-1 protein in silkworm. It was upregulated at both the mRNA level and the proteomic level and may play an important role in regulating the autophagy and apoptosis (especially apoptosis) induced by BmCPV infection. This was the first report using an iTRAQ approach to analyze proteomes of the silkworm midgut against BmCPV infection, which contributes to understanding the defense mechanisms of silkworm midgut to virus infection. The domesticated silkworm, Bombyx mori, is renowned for silk production as well as being a traditional lepidopteron model insect served as a subject for morphological, genetic, physiological, and developmental studies. Bombyx mori cytoplasmic polyhedrosis

  2. Highly efficient multiplex targeted mutagenesis and genomic structure variation in Bombyx mori cells using CRISPR/Cas9.

    PubMed

    Liu, Yuanyuan; Ma, Sanyuan; Wang, Xiaogang; Chang, Jiasong; Gao, Jie; Shi, Run; Zhang, Jianduo; Lu, Wei; Liu, Yue; Zhao, Ping; Xia, Qingyou

    2014-06-01

    Bombyx mori is an economically important insect and a model organism for studying lepidopteran and arthropod biology. Using a highly efficient CRISPR/Cas9 system, we showed that this system could mediate highly efficient targeted genome editing of a single gene locus, large chromosomal deletion or inversion, and also multiplex genome editing of 6 genes simultaneously in BmNs cell line derived from B. mori. The simplicity and high efficiency of our system provide unprecedented possibilities for researchers to implement precise and sophisticated manipulation of a chosen B. mori gene in BmNs cells easily in a limited time course, and perhaps new opportunities for functional genomics of B. mori and other lepidopteran insects.

  3. Determination of intermolecular distance for a model peptide of Bombyx mori silk fibroin, GAGAG, with rotational echo double resonance.

    PubMed

    Kameda, Tsunenori; Nakazawa, Yasumoto; Kazuhara, Junko; Yamane, Tsutomu; Asakura, Tetsuo

    2002-07-05

    Rotational echo double resonance NMR spectroscopy is applied for the determination of the distance of intermolecular chains of pentapeptide, GAGAG (G: Gly, A: Ala), a model typical of the crystalline domain in Bombyx mori silk fibroin. 1:4 mixture of G[1-(13)C]AGAG and GAG[(15)N]AG with antiparallel beta-sheet structure was used to determine the distance of intermolecular hydrogen bonding between adjacent molecules within pleated sheet and the (13)C-(15)N interatomic distance was determined to be 4.3 A. On the other hand, 1:4 mixture of GAG[1-(13)C]AG and GAG[(15)N]AG gave information on the interpleated sheet arrangement. When we assumed the same distances between two interpleated sheets, the distance was calculated to be 5.3 A and the angle (15)N-(13)C-(15)N was 180 degrees. Copyright 2002 Wiley Periodicals, Inc.

  4. Bombyx mori Transcription Factors: Genome-Wide Identification, Expression Profiles and Response to Pathogens by Microarray Analysis

    PubMed Central

    Huang, Lulin; Cheng, Tingcai; Xu, Pingzhen; Fang, Ting; Xia, Qingyou

    2012-01-01

    Transcription factors are present in all living organisms, and play vital roles in a wide range of biological processes. Studies of transcription factors will help reveal the complex regulation mechanism of organisms. So far, hundreds of domains have been identified that show transcription factor activity. Here, 281 reported transcription factor domains were used as seeds to search the transcription factors in genomes of Bombyx mori L. (Lepidoptera: Bombycidae) and four other model insects. Overall, 666 transcription factors including 36 basal factors and 630 other factors were identified in B. mori genome, which accounted for 4.56% of its genome. The silkworm transcription factors' expression profiles were investigated in relation to multiple tissues, developmental stages, sexual dimorphism, and responses to oral infection by pathogens and direct bacterial injection. These all provided rich clues for revealing the transcriptional regulation mechanism of silkworm organ differentiation, growth and development, sexual dimorphism, and response to pathogen infection. PMID:22943524

  5. The BRO proteins of Bombyx mori nucleopolyhedrovirus are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway

    SciTech Connect

    Kang, Won Kyung . E-mail: wkkang@riken.jp; Kurihara, Masaaki . E-mail: mkuri@riken.jp; Matsumoto, Shogo . E-mail: smatsu@riken.jp

    2006-06-20

    The BRO proteins of Bombyx mori nucleopolyhedrovirus (BmNPV) display a biphasic pattern of intracellular localization during infection. At early times, they reside in the nucleus but then show both cytoplasmic and nuclear localization as the infection proceeds. Therefore, we examined the possibility of nuclear export. Using inhibitors, we reveal that BmNPV BRO proteins shuttle between the nucleus and cytoplasm. Mutations on the leucine-rich region of BRO proteins resulted in nuclear accumulation of transiently expressed proteins, suggesting that this region functions as a CRM1-dependent nuclear export signal (NES). On the contrary, mutant BRO-D with an altered NES did not show nuclear accumulation in infected cells, although protein production seemed to be reduced. RT-PCR analysis showed that the lower level of protein production was due to a reduction in RNA synthesis. Taken together, our results suggest that BRO proteins are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway.

  6. Differentially Expressed Genes in the Cuticle and Hemolymph of the Silkworm, Bombyx mori, Injected with the Fungus Beauveria bassiana

    PubMed Central

    Hou, Cheng-Xiang; Qin, Guang-Xing; Liu, Ting; Mei, Xing-Lin; Li, Bing; Shen, Zhong-Yuan; Guo, Xi-Jie

    2013-01-01

    The most important pathogenic fungus of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), is Beauveria bassiana (Balsamo-Crivelli ) Vuillemin (Hypocreales: Clavicipitaceae), which causes significant damage to sericulture production. Therefore, diagnosing fungal disease and developing new control measures are crucial to silk production. To better understand the responsive and interactive mechanisms between the host silkworm and this fungus, variations in silkworm gene expression were investigated using the suppression subtractive hybridization method following the injection of B. bassiana conidia. Two cDNA libraries were constructed, and 140 cDNA clones were isolated. Of the 50 differentially expressed genes identified, 45 (112 clones) were identified in the forward library, and 5 (28 clones) were identified in the reverse library. Expression profiling of six of these genes by quantitative polymerase chain reaction (qPCR) verified that they were induced by the fungal challenge. The present study provides insight into the interaction between lepidopteran insects and pathogenic fungi. PMID:24794288

  7. A novel granulocyte-specific α integrin is essential for cellular immunity in the silkworm Bombyx mori.

    PubMed

    Zhang, Kui; Tan, Juan; Xu, Man; Su, Jingjing; Hu, Renjian; Chen, Yibiao; Xuan, Fan; Yang, Rui; Cui, Hongjuan

    2014-12-01

    Haemocytes play crucial roles in immune responses and survival in insects. Specific cell markers have proven effective in clarifying the function and haematopoiesis of haemocytes. The silkworm Bombyx mori is a good model for studying insect haemocytes; however, little is known about haemocyte-specific markers or their functions in silkworm. In this study, we identified the α subunit of integrin, BmintegrinαPS3, as being specifically and highly expressed in silkworm haemocytes. Immunofluorescence analysis validated the specificity of BmintegrinαPS3 in larval granulocytes. Further analyses indicated that haemocytes dispersed from haematopoietic organs (HPOs) into the circulating haemolymph could differentiate into granulocytes. In addition, the processes of encapsulation and phagocytosis were controlled by larval granulocytes. Our work demonstrated that BmintegrinαPS3 could be used as a specific marker for granulocytes and could be applied to future molecular cell biology studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. The BRO proteins of Bombyx mori nucleopolyhedrovirus are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway.

    PubMed

    Kang, WonKyung; Kurihara, Masaaki; Matsumoto, Shogo

    2006-06-20

    The BRO proteins of Bombyx mori nucleopolyhedrovirus (BmNPV) display a biphasic pattern of intracellular localization during infection. At early times, they reside in the nucleus but then show both cytoplasmic and nuclear localization as the infection proceeds. Therefore, we examined the possibility of nuclear export. Using inhibitors, we reveal that BmNPV BRO proteins shuttle between the nucleus and cytoplasm. Mutations on the leucine-rich region of BRO proteins resulted in nuclear accumulation of transiently expressed proteins, suggesting that this region functions as a CRM1-dependent nuclear export signal (NES). On the contrary, mutant BRO-D with an altered NES did not show nuclear accumulation in infected cells, although protein production seemed to be reduced. RT-PCR analysis showed that the lower level of protein production was due to a reduction in RNA synthesis. Taken together, our results suggest that BRO proteins are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway.

  9. Occurrence of ommochrome-containing pigment granules in the central nervous system of the silkworm, Bombyx mori.

    PubMed

    Sawada, H; Nakagoshi, M; Mase, K; Yamamoto, T

    2000-03-01

    Dark-red pigment granules were found in the brain and ganglion of the normal strain of the silkworm, Bombyx mori, by light microscopy. No other pigmentation was seen in the brain or ganglia. Electron microscopy showed that the granules were electron-dense. The granules were similar to the ommochrome-containing pigment granules that are present in the epidermal cells of the quail mutant, as previously reported. The pigment in the larval central nervous system (CNS) of the normal silkworm was identical to the ommin standard with respect to the absorption spectrum, the infrared spectrum, and the Rf value in thin-layer chromatography (TLC). After acid hydrolysis of the pigment, 3-hydroxykynurenine was detected by TLC. The pigment granules in the CNS contained mainly ommin. An ommochrome-binding protein was also detected in the CNS by in vitro binding studies and Western blotting. The ommochrome granules may have an important function in the CNS of the silkworm.

  10. Shotgun proteomic analysis of the Bombyx mori anterior silk gland: An insight into the biosynthetic fiber spinning process.

    PubMed

    Yi, Qiying; Zhao, Ping; Wang, Xin; Zou, Yong; Zhong, Xiaowu; Wang, Chen; Xiang, Zhonghuai; Xia, Qing-You

    2013-09-01

    The Bombyx mori anterior silk gland (ASG) is a natural fiber manipulator for the material provided by the middle and posterior silk glands. In view of the significant role of the ASG in the liquid-crystal spinning process, a shotgun proteomics approach was taken to study the relationship between the function of proteins in the silkworm ASG and the spinning mechanism. A total of 1132 proteins with 7647 unique peptides were identified in the ASG dataset including some involved in the cuticle, ion transportation, energy metabolism, and apoptosis. Two putative cuticle-specific proteins were highly and specifically expressed in the ASG; therefore, the ASG dataset could provide clues for comprehensive understanding of the natural silk spinning mechanism in the silkworm. All MS data have been deposited in the ProteomeXchange with identifier PXD000090. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. A novel third chromosomal locus controls susceptibility to Autographa californica multiple nucleopolyhedrovirus in the silkworm, Bombyx mori.

    PubMed

    Xu, Jian; Kusakabe, Takahiro; Yamamoto, Kimiko; Suetsugu, Yoshitaka; Mon, Hiroaki; Li, Zhiqing; Zhu, Li; Iiyama, Kazuhiro; Banno, Yutaka; Yoshimura, Kaito; Lee, Jae Man

    2014-04-01

    Baculovirus demonstrates specific infection spectrums and thus one certain host exhibits particular response to single baculovirus isolate. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is considered to be not an innate pathogen to Bombyx mori, but some silkworm strains have been identified to be permissive to AcMNPV, indicating the positive or negative involvement of certain host factors in baculovirus replications in vivo. To provide a fundamental knowledge of this process, we performed large-scale screening to investigate the responses of 448 silkworm strains against recombinant AcMNPV inoculation. By genetic analysis between permissive and resistant strains identified, we further confirmed that a potential corresponding locus on chromosome 3 regulates host responses to AcMNPV in silkworm. Additionally, we found that it is available for AcMNPV-silkworm baculovirus expression vector system to produce proteins of interest.

  12. Microscopy based studies on the interaction of bio-based silver nanoparticles with Bombyx mori Nuclear Polyhedrosis virus.

    PubMed

    Tamilselvan, Selvaraj; Ashokkumar, Thirunavukkarasu; Govindaraju, Kasivelu

    2017-04-01

    In the present investigation, silver nanoparticles (AgNPs) interactions with Bombyx mori Nuclear Polyhedrosis virus (BmNPV) were characterized using High-Resolution Scanning Electron Microscopy (HR-SEM), Energy Dispersive X-ray Analysis (EDAX), Transmission Electron Microscopy (TEM), Atomic Force Microcopy (AFM) and Confocal Microscope (CM). HR-SEM study reveals that the biosynthesized AgNPs have interacted with BmNPV and were found on the surface. TEM micrographs of normal and viral polyhedra treated with AgNPs showed that the nanoparticles were accumulated in the membrane and it was noted that some of the AgNPs successfully penetrated the membrane by reaching the capsid of BmNPV. AFM and confocal microscopy studies reveal that the disruption in the shell membrane tends to lose its stability due to exposure of AgNPs to BmNPV.

  13. A histochemical study of the posterior silk glands of Bombyx mori during metamorphosis from larvae to pupae using frozen sections.

    PubMed

    Kawamoto, K; Kawamoto, T; Shiba, H; Hosono, K

    2014-02-01

    The fine structures of the whole bodies and the posterior silk glands of Bombyx mori during metamorphosis from larvae to pupae in the cocoon were preserved virtually without damage when frozen sections were prepared using an adhesive plastic film. We used frozen sections for histochemical and enzyme histochemistry to characterize the metamorphosis of the posterior silk glands. Frozen sections were stained with DAPI to observe nuclear changes, examined using the TUNEL method to detect DNA fragments, and investigated using in situ hybridization to detect B. mori caspase expression. Both DNA fragments and expression of B. mori caspase increased with progressing metamorphosis. The degeneration of the posterior silk gland during metamorphosis appears to be an apoptotic event.

  14. Antioxidative properties of 4-methylumbelliferone are related to antibacterial activity in the silkworm (Bombyx mori) digestive tract.

    PubMed

    Fang, Yan; Wang, Hua; Zhu, Wenjuan; Wang, Lu; Liu, Hengjiang; Xu, Xu; Yin, Weimin; Sima, Yanghu; Xu, Shiqing

    2014-08-01

    Umbelliferones have gained significant attention due to their tumor-inhibitory effects in vitro. This study was undertaken to examine the impact of umbelliferones in an invertebrate model organism, Bombyx mori, to assess the underlying antimicrobial activities via antioxidation in vivo. Oral administration of 4 mM 4-methylumbelliferone (4-MU), a model umbelliferone drug, in B. Mori larvae caused a rapid increase in reactive oxygen species, such as hydrogen peroxide (H2O2) and antimicrobial activity in the digestive tract. In addition, a significant increase in total antioxidant capacity as well as superoxide anion radical-inhibiting activity and reduced glutathione were detected. The antioxidant defense system was activated following induction of H2O2, resulting in a significant rise in catalase (50-66 %) and glutathione peroxidase (175 %) activities, which were helpful in defending digestive tract cells against oxidative injury. These results help in understanding the anticancer mechanism of 4-MU based on its antioxidation in organisms.

  15. Rapid Detection of Infectious Flacherie Virus of the Silkworm, Bombyx mori, using RT-PCR and Nested PCR

    PubMed Central

    Vootla, Shyam Kumar; Lu, Xing Meng; Kari, Neetha; Gadwala, Mallikarjun; Lu, Qineng

    2013-01-01

    In this study, a method for detection of an ssRNA viral pathogen that causes viral flacherie in the silkworm, Bombyx mori (L.) (Lepidoptera: Bombycidae), was used for the detection of B. mori infectious flacherie virus (BmIFV). A combination of nested and reverse transcriptase polymerase chain reaction was used for detection. Although BmIFV has been reported in almost all the sericultural regions of the world, there had been no reports of BmIFV incidence in India. Therefore, the confirmation of the presence of BmIFV in Karnataka, India, is of great significance. The present method is advantageous because it can be used to detect the virus by using samples from infected midgut tissues, thus simplifying and avoiding laborious genome isolation procedures. This method could help in early detection of BmIFV disease pathogens and help reduce crop losses. PMID:24785655

  16. Cerium chloride improves protein and carbohydrate metabolism of fifth-instar larvae of Bombyx mori under phoxim toxicity.

    PubMed

    Li, Bing; Xie, Yi; Cheng, Zhe; Cheng, Jie; Hu, Rengping; Sang, Xuezi; Gui, Suxin; Sun, Qingqing; Gong, Xiaolan; Cui, Yaling; Shen, Weide; Hong, Fashui

    2012-12-01

    The organophosphorus pesticide poisoning of the silkworm Bombyx mori is one of the major events causing serious damage to sericulture. Added low-dose rare earths are demonstrated to increase resistance in animals. However, very little is known about whether or not added CeCl₃ can increase resistance of silkworm to phoxim poisoning. The present findings suggested that added CeCl₃ to mulberry leaves markedly increased contents of protein, glucose and pyruvate, and carbohydrate metabolism-related enzyme activities, including lactate dehydrogenase, succinate dehydrogenase and malate dehydrogenase, and attenuated free amino acids, urea, uric acid and lactate levels and inhibited the protein metabolism-related enzymes activities, such as protease, alanine aminotransferase and aspartate aminotransferase in the haemolymph of B. mori, under phoxim toxicity. These findings suggest that added CeCl₃ may improve protein and carbohydrate metabolisms, thus leading to increases of growth and survival rate of B. mori under phoxim stress.

  17. Differentially expressed genes in the cuticle and hemolymph of the silkworm, Bombyx mori, injected with the fungus Beauveria bassiana.

    PubMed

    Hou, Cheng-Xiang; Qin, Guang-Xing; Liu, Ting; Mei, Xing-Lin; Li, Bing; Shen, Zhong-Yuan; Guo, Xi-Jie

    2013-01-01

    The most important pathogenic fungus of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), is Beauveria bassiana (Balsamo-Crivelli ) Vuillemin (Hypocreales: Clavicipitaceae), which causes significant damage to sericulture production. Therefore, diagnosing fungal disease and developing new control measures are crucial to silk production. To better understand the responsive and interactive mechanisms between the host silkworm and this fungus, variations in silkworm gene expression were investigated using the suppression subtractive hybridization method following the injection of B. bassiana conidia. Two cDNA libraries were constructed, and 140 cDNA clones were isolated. Of the 50 differentially expressed genes identified, 45 (112 clones) were identified in the forward library, and 5 (28 clones) were identified in the reverse library. Expression profiling of six of these genes by quantitative polymerase chain reaction (qPCR) verified that they were induced by the fungal challenge. The present study provides insight into the interaction between lepidopteran insects and pathogenic fungi.

  18. Juvenile Hormone Analogues, Methoprene and Fenoxycarb Dose-Dependently Enhance Certain Enzyme Activities in the Silkworm Bombyx Mori (L)

    PubMed Central

    Mamatha, Devi M.; Kanji, Vijaya K.; Cohly, Hari H.P.; Rao, M. Rajeswara

    2008-01-01

    Use of Juvenile Hormone Analogues (JHA) in sericulture practices has been shown to boost good cocoon yield; their effect has been determined to be dose-dependent. We studied the impact of low doses of JHA compounds such as methoprene and fenoxycarb on selected key enzymatic activities of the silkworm Bombyx mori. Methoprene and fenoxycarb at doses of 1.0 μg and 3.0fg/larvae/48 hours showed enhancement of the 5th instar B. mori larval muscle and silkgland protease, aspartate aminotransaminase (AAT) and alanine aminotransaminase (ALAT), adenosine triphosphate synthase (ATPase) and cytochrome-c-oxidase (CCO) activity levels, indicating an upsurge in the overall oxidative metabolism of the B.mori larval tissues. PMID:18678927

  19. Juvenile Hormone Analogues, methoprene and fenoxycarb dose-dependently enhance certain enzyme activities in the silkworm Bombyx mori (L).

    PubMed

    Mamatha, Devi M; Kanji, Vijaya K; Cohly, Hari H P; Rao, M Rajeswara

    2008-06-01

    Use of Juvenile Hormone Analogues (JHA) in sericulture practices has been shown to boost good cocoon yield; their effect has been determined to be dose-dependent. We studied the impact of low doses of JHA compounds such as methoprene and fenoxycarb on selected key enzymatic activities of the silkworm Bombyx mori. Methoprene and fenoxycarb at doses of 1.0 microg and 3.0 fg/larvae/48 hours showed enhancement of the 5th instar B. mori larval muscle and silkgland protease, aspartate aminotransaminase (AAT) and alanine aminotransaminase (ALAT), adenosine triphosphate synthase (ATPase) and cytochrome-c-oxidase (CCO) activity levels, indicating an upsurge in the overall oxidative metabolism of the B.mori larval tissues.

  20. The ecdysteroidogenic P450 Cyp302a1/disembodied from the silkworm, Bombyx mori, is transcriptionally regulated by prothoracicotropic hormone.

    PubMed

    Niwa, R; Sakudoh, T; Namiki, T; Saida, K; Fujimoto, Y; Kataoka, H

    2005-10-01

    During larval and pupal development of insects, ecdysone is synthesized in the prothoracic gland (PG). Although several Drosophila genes, including Halloween P450 genes, are known to be important for ecdysteroidogenesis in PG, little is known of the ecdysteroidogenic genes in other insects. Here we report on Cyp302a1/disembodied (dib-Bm), one of the Halloween P450s in the silkworm Bombyx mori that is a carbon-22 hydroxylase. dib-Bm is predominantly expressed in PG and its developmental expression profile is correlated with a change in the ecdysteroid titre in the haemolymph. Furthermore, dib-Bm expression in cultured PGs is significantly induced by treatment with prothoracicotropic hormone. This is the first report on the transcriptional induction of a steroidogenic gene by the tropic hormone in insects.

  1. Genetic mapping of a food preference gene in the silkworm, Bombyx mori, using restriction fragment length polymorphisms (RFLPs).

    PubMed

    Mase, Keisuke; Iizuka, Tetsuya; Yamamoto, Toshio; Okada, Eiji; Hara, Wajirou

    2007-06-01

    The domesticated silkworm, Bombyx mori, has strict food preferences and grows by feeding on mulberry leaves. However, "Sawa-J", an abnormal feeding habit strain selected from the genetic stock, feeds on an artificial diet without mulberry leaf powder. In this study, the food preference gene in Sawa-J was genetically identified using restriction fragment length polymorphisms (RFLPs) of a cDNA clone on each linkage group. Taking advantage of a lack of genetic recombination in females, reciprocal backcrossed F1 (BC1) progenies were independently prepared using a non-feeding strain, C108, as a mating partner of Sawa-J. Our results of linkage analysis and mapping proved that the feeding behavior is primarily controlled by a major recessive gene mapped at 20.2 cM on RFLP linkage group 9 (RFLG9), and clone e73 at a distance of 4.2 cM was found as the first linked molecular marker.

  2. Crystal structure of Bombyx mori lipoprotein 6: comparative structural analysis of the 30-kDa lipoprotein family.

    PubMed

    Pietrzyk, Agnieszka J; Bujacz, Anna; Łochynska, Malgorzata; Jaskolski, Mariusz; Bujacz, Grzegorz

    2014-01-01

    The 30-kDa lipoprotein (LP) family of mulberry silkworm comprises major hemolymph proteins specific to the fifth instar larvae. The family consists of 46 members, 24 of which are referred to as typical 30-kDa LPs. To date, two crystal structures of 30-kDa LPs from Bombyx mori have been described (Bmlp3 and Bmlp7). Here, we present the crystal structure of Bmlp6, another 30-kDa LP member. Bmlp6 is comprised of two domains characteristic of this family, the VHS-type N-terminal domain and β-trefoil C-terminal domain. The structures of the three 30-kDa LPs have been compared and a number of differences are noted, including loop conformation, the surface electrostatic potential, and the potential binding cavities. We discuss the observed structural differences in the light of the potential different roles of the particular 30-kDa LP members in silkworm physiology.

  3. Cloning and Expression Analysis of the Bombyx mori α-amylase Gene (Amy) from the Indigenous Thai Silkworm Strain, Nanglai

    PubMed Central

    Ngernyuang, Nipaporn; Kobayashi, Isao; Promboon, Amornrat; Ratanapo, Sunanta; Tamura, Toshiki; Ngernsiri, Lertluk

    2011-01-01

    α-Amylase is a common enzyme for hydrolyzing starch. In the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), α-amylase is found in both digestive fluid and hemolymph. Here, the complete genomic sequence of the Amy gene encoding α-amylase from a local Thai silkworm, the Nanglai strain, was obtained. This gene was 7981 bp long with 9 exons. The full length Amy cDNA sequence was 1749 bp containing a 1503 bp open reading frame. The ORF encoded 500 amino acid residues. The deduced protein showed 81–54% identity to other insect α-amylases and more than 50% identity to mammalian enzymes. Southern blot analysis revealed that in the Nanglai strain Amy is a single-copy gene. RT- PCR showed that Amy was transcribed only in the foregut. Transgenic B. mori also showed that the Amy promoter activates expression of the transgene only in the foregut. PMID:21529256

  4. Silk gland-specific proteinase inhibitor serpin16 from the Bombyx mori shows cysteine proteinase inhibitory activity.

    PubMed

    Guo, Peng-Chao; Dong, Zhaoming; Xiao, Li; Li, Tao; Zhang, Yan; He, Huawei; Xia, Qingyou; Zhao, Ping

    2015-01-30

    Serpins (serine proteinase inhibitors) are widely distributed in different species and are well known for their inhibitory activities towards serine proteinases. Here, we report the functional characterization of Bombyx mori serpin16. Expression analysis showed that serpin16 was specifically expressed at high levels in the silk gland at both the transcriptional and translational levels. Moreover, homology modeling and multi-sequence alignment suggested that serpin16 had a canonical serpin fold, but it contained a unique reactive center loop, which was obviously shorter than that of typical serpins. Inhibitory activity analyses revealed that the target proteinase of serpin18 is a cysteine proteinase, rather than a serine proteinase. Furthermore, a Michaelis complex model of serpin16 with its target proteinase was constructed to explain the structural basis of how serpin16 recognizes the cysteine proteinase and its target specificity.

  5. Effects of Titanium Dioxide Nanoparticles on the Synthesis of Fibroin in Silkworm (Bombyx mori).

    PubMed

    Ni, Min; Li, FanChi; Tian, JiangHai; Hu, JingSheng; Zhang, Hua; Xu, KaiZun; Wang, BinBin; Li, YangYang; Shen, WeiDe; Li, Bing

    2015-08-01

    Silkworm (Bombyx mori) is an economically important insect, and its silk production capacity largely depends on its ability to synthesize fibroin. While breeding of B. mori varieties has been a key strategy to improve silk production, little improvement of B. mori silk production has been achieved to date. As a result, the development of sericulture economy has not progressed well, pointing to the need of new ways for improvement of B. mori silk production. Titanium dioxide nanoparticles (TiO2 NPs), a food additive widely used for livestock, have been shown to promote animal growth and increase the protein synthesis in animals. However, no studies on effect of TiO2 NPs on fibroin synthesis in B. mori have been available. In this study, the differential expression profiles of genes and proteins in the silk gland of B. mori fed without or with TiO2 NPs (5 μg ml(-1)) were analyzed and compared using digital gene expression (DGE), reverse transcription quantitative polymerase chain reaction (RT-qPCR), semi-qPCR, and Western blot analysis. The effects of TiO2 NPs feeding on the activity of proteases in the midgut and the synthesis and transportation of amino acids in hemolymph were also investigated. DGE analyses showed that among a total of 4,741 genes detected, 306 genes were differentially expressed after the TiO2 NPs feeding, of which 137 genes were upregulated whereas 169 genes were downregulated. 106 genes were shown to be involved in fibroin synthesis, of which 97 genes, including those encoding cuticular protein glycine-rich 10, serine protease inhibitor 28, aspartate aminotransferase, lysyl-tRNA synthetase, and splicing factor arginine/serine-rich 6, and silk gland factor-1 (SGF-1), were upregulated with the maximum induction of 8.52-folds, whereas nine genes, including those encoding aspartylglucosaminidase, the cathepsin L in Tribolium castaneum, and similar to SPRY domain-containing SOCS box protein 3, were downregulated with the maximum reduction of 8

  6. Novel Infection System of Recombinant BmBDV DNA into BmN Cells of Silkworm, Bombyx mori.

    PubMed

    Guo, Rui; Cao, Guangli; Zhu, Yuexiong; Kumar, Dhiraj; Xue, Renyu; Lu, Yahong; Hu, Xiaolong; Gong, Chengliang

    2016-10-01

    Bombyx mori bidensovirus (BmBDV) was previously termed as Bombyx mori densovirus type 2 and later it was reclassified in the new genus bidensovirus of the new family Bidnaviridae. The genome of BmBDV Zhenjiang isolate (BmBDV-Z) consists of two non-homologous single-stranded linear DNA molecules VD1 and VD2 which are encapsidated into separate virion. To investigate the infectivity of BmBDV DNA, recombinant plasmids pGEM-VD1 inserted with VD1 genome were transfected into the BmN cells of silkworm. Structural proteins of BmBDV were detected with Western blot and immunofluorescence assay, which indicates pGEM-VD1 replicated in the transfected BmN cells and viral proteins were also expressed. Through TEM observation, we identified about 20 nm BmBDV-like viral particles, which confirmed that BmBDV can be generated after transfection. Subsequently, a recombinant baculovirus BmBac-VD1 inserted with VD1 genome was constructed. Results of Western blot and immunofluorescence assay indicated that viral structural proteins of BmBDV were expressed in the BmBac-VD1-infected cells. Baculiform and spherical virions were also observed in infected cells by TEM, and two kinds of virions were separated. However, results of molecular biological detection revealed that infectious sequence from BmBac-VD1 was packaged within spherical virion. Therefore, we suggested that vector inserted with BmBDV genomic DNA showed infectivity, and BmBDV-like viral particles packaging recombinant DNA can be produced in the cultured BmN cells. Outcome of our current research provided not only a new method of infection to explore the gene function of BmBDV in vitro but also a protocol to facilitate development of more effective new-type pesticides.

  7. 20-Hydroxyecdysone (20E) Primary Response Gene E93 Modulates 20E Signaling to Promote Bombyx Larval-Pupal Metamorphosis*

    PubMed Central

    Liu, Xi; Dai, Fangyin; Guo, Enen; Li, Kang; Ma, Li; Tian, Ling; Cao, Yang; Zhang, Guozheng; Palli, Subba R.; Li, Sheng

    2015-01-01

    As revealed in a previous microarray study to identify genes regulated by 20-hydroxyecdysone (20E) and juvenile hormone (JH) in the silkworm, Bombyx mori, E93 expression in the fat body was markedly low prior to the wandering stage but abundant during larval-pupal metamorphosis. Induced by 20E and suppressed by JH, E93 expression follows this developmental profile in multiple silkworm alleles. The reduction of E93 expression by RNAi disrupted 20E signaling and the 20E-induced autophagy, caspase activity, and cell dissociation in the fat body. Reducing E93 expression also decreased the expression of the 20E-induced pupal-specific cuticle protein genes and prevented growth and differentiation of the wing discs. Importantly, the two HTH domains in E93 are critical for inducing the expression of a subset of 20E response genes, including EcR, USP, E74, Br-C, and Atg1. By contrast, the LLQHLL and PLDLSAK motifs in E93 inhibit its transcriptional activity. E93 binds to the EcR-USP complex via a physical association with USP through its LLQHLL motif; and this association is enhanced by 20E-induced EcR-USP interaction, which attenuates the transcriptional activity of E93. E93 acts through the two HTH domains to bind to GAGA-containing motifs present in the Atg1 promoter region for inducing gene expression. In conclusion, E93 transcriptionally modulates 20E signaling to promote Bombyx larval-pupal metamorphosis. PMID:26378227

  8. 20-Hydroxyecdysone (20E) Primary Response Gene E93 Modulates 20E Signaling to Promote Bombyx Larval-Pupal Metamorphosis.

    PubMed

    Liu, Xi; Dai, Fangyin; Guo, Enen; Li, Kang; Ma, Li; Tian, Ling; Cao, Yang; Zhang, Guozheng; Palli, Subba R; Li, Sheng

    2015-11-06

    As revealed in a previous microarray study to identify genes regulated by 20-hydroxyecdysone (20E) and juvenile hormone (JH) in the silkworm, Bombyx mori, E93 expression in the fat body was markedly low prior to the wandering stage but abundant during larval-pupal metamorphosis. Induced by 20E and suppressed by JH, E93 expression follows this developmental profile in multiple silkworm alleles. The reduction of E93 expression by RNAi disrupted 20E signaling and the 20E-induced autophagy, caspase activity, and cell dissociation in the fat body. Reducing E93 expression also decreased the expression of the 20E-induced pupal-specific cuticle protein genes and prevented growth and differentiation of the wing discs. Importantly, the two HTH domains in E93 are critical for inducing the expression of a subset of 20E response genes, including EcR, USP, E74, Br-C, and Atg1. By contrast, the LLQHLL and PLDLSAK motifs in E93 inhibit its transcriptional activity. E93 binds to the EcR-USP complex via a physical association with USP through its LLQHLL motif; and this association is enhanced by 20E-induced EcR-USP interaction, which attenuates the transcriptional activity of E93. E93 acts through the two HTH domains to bind to GAGA-containing motifs present in the Atg1 promoter region for inducing gene expression. In conclusion, E93 transcriptionally modulates 20E signaling to promote Bombyx larval-pupal metamorphosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Expression profiling of Bombyx mori gloverin2 gene and its synergistic antifungal effect with cecropin A against Beauveria bassiana.

    PubMed

    Lü, Dingding; Geng, Tao; Hou, Chengxiang; Qin, Guangxing; Gao, Kun; Guo, Xijie

    2017-02-05

    Gloverin2 is a cationic and glycine-rich antimicrobial peptide whose expression can be induced in fat body of silkworm (Bombyx mori) larvae exposed to bacteria. The purpose of this study is to identify the roles of Bombyx mori gloverin2 (Bmgloverin2) during entomopathogenic fungus Beauveria bassiana infection. Fluorescent quantitative real-time PCR analysis indicated that the relative expression level of Bmgloverin2 gene was up-regulated in the silkworm larvae infected by B. bassiana. The cDNA of Bmgloverin2 was cloned from the silkworm by RT-PCR and the DNA segment of the Bmgloverin2 peptide (without signal peptide sequence) was inserted into pCzn1 expression plasmid and expressed in E. coli ArcticExpress (DE3). SDS-PAGE results revealed that soluble recombinant Bmgloverin2 was successfully expressed and purified. Polyclonal antibody against the Bmgloverin2 was successfully produced with the expressed recombinant protein. Western blot analysis indicated that Bmgloverin2 could be detected in the fat body of silkworm larvae infected with B. bassiana, suggesting that the expression of Bmgloverin2 could be induced by B. bassiana infection in silkworm. Antifungal assays indicated that the Bmgloverin2 had a synergistic antifungal activity with B. mori cecropin A (BmCecA) to entomopathogenic fungus B. bassiana both in vitro and in vivo in the silkworm larvae. This is the first report that Bmgloverin2 exhibits synergistic effect with BmCecA in antifungal activity against B. bassiana. The study demonstrates that Bmgloverin2 is an antifungal protein which plays an important role in synergistic antifungal activity with other antimicrobial peptide in silkworm.

  10. Structural analysis of TRAS1, a novel family of telomeric repeat-associated retrotransposons in the silkworm, Bombyx mori.

    PubMed Central

    Okazaki, S; Ishikawa, H; Fujiwara, H

    1995-01-01

    We characterized TRAS1, a retrotransposable element which was inserted into the telomeric repetitive sequence (CCTAA)n of the silkworm, Bombyx mori. The complete sequence of TRAS1, a stretch of 7.8 kb with a poly(A) tract at the 3' end, was determined. No long terminal repeat (LTR) was found at the termini of the element. TRAS1 contains gag- and pol-like open reading frames (ORFs) which are similar to those of non-LTR retrotransposons. The two ORFs overlap but are one nucleotide out of frame (+1 frameshift). Most of the approximately 250 copies of TRAS1 elements in the genome were highly conserved in the structure. Chromosomal in situ hybridization showed that TRAS1 elements are clustered at the telomeres of Bombyx chromosomes. A phylogenetic analysis using the amino acid sequence of the reverse transcriptase domain within the pol-like ORF revealed that TRAS1 falls into one lineage with R1, which is a family of non-LTR retrotransposons inserted into the same site within the 28S ribosomal DNA unit in most insects. TRAS1 may have been derived from R1 and changed the target specificity so that TRAS1 inserts into the telomeric repetitive sequence (CCTAA)n. Southern hybridization and Bal 31 exonuclease analyses showed that TRAS1 elements are clustered proximal to the terminal long tract of (CCTAA)n. TRAS1 is a novel family of non-LTR retrotransposons which are inserted into the telomeric repetitive sequences as target sites. PMID:7623845

  11. Differential expression of the Bombyx mori diapause-termination timer gene Ea4 in diapause-inducing temperature and photoperiod.

    PubMed

    Xu, Li; Liang, Hui; Niu, Yanshan; Wang, Ying; Sima, Yanghu; Xu, Shiqing

    2012-03-01

    Diapause in Bombyx mori eggs is induced by temperature and photoperiod at the stage of embryonic development in the maternal generation. In those diapause eggs, Esterase-A4 is suggested to serve as a diapause-termination timer (TIME-EA4), because its ATPase activity shows an interval-timer elevation after acid treatment or chilling of eggs to break diapause. To clarify whether the timed ATPase activity of TIME-EA4 is related to its gene (Ea4) expression, we analyzed Ea4 mRNA of eggs in diapause-inducing environmental conditions. Reverse transcription PCR (RT-PCR) analysis showed that the level of Ea4 mRNA was lower in 15DD than in 25LL (P < 0.01) or in 20LD (P < 0.01) but did not oscillate when photoperiod and temperature periodically oscillated (P > 0.05). Furthermore, expressed sequence tag profile and gene microarray analysis demonstrated that Ea4 showed stage-specific and tissue-specific expression during postembryonic stages, high Ea4 mRNA in the spinning and eclosion stages, and in integument and head, but low in gonads of fifth-instar day-3 larvae. Then we analyzed the relationship between TIME-EA4 ATPase activities and Ea4 gene expression. The ATPase activities in diapause eggs laid by the resultant adults transiently elevated after treatments to break diapause, that is, at 1.5 h after common-acid treatment and day 13 after chilling at 5°C. However, these elevations of enzyme activities were not accompanied by any increases in Ea4 mRNA levels. In conclusion, the termination of the Bombyx embryonic diapause is related to TIME-EA4 but not to its gene expression. © 2012 Wiley Periodicals, Inc.

  12. An investigation and evaluation on species and characteristics of pathogenic microorganisms in Chinese local hospital settings.

    PubMed

    Chen, Liang; Lü, Xiaoli; Cao, Weiping; Zhang, Chunxia; Xu, Rongfa; Meng, Xu; Chen, Keping

    2015-12-01

    There are currently great concerns about the level of bacterial contamination in hospitals, as well as resistance to antimicrobial agents. The species and characteristics of microbes in Chinese hospitals are closely related to healthcare safety and the prevention and control of infections. However, data on the exposure of patients to microbes in Chinese hospitals are limited. The present study investigated the genera of microorganisms in Chinese hospitals. We evaluated their characteristics, such as antibiotic susceptibility, tolerance to disinfectants, and toxicity, using silkworms (Bombyx mori) as an animal model. Twenty-six distinct bacterial strains were isolated, and their genera were determined by sequencing their 16S rDNA regions. Twenty-five strains were resistant to one or more antibiotics, and six strains were resistant to multiple antibiotics. The results of minimal inhibitory concentration testing showed that eight strains were resistant to a chlorine-containing disinfectant, and 12 strains were resistant to Povidone-iodine. Following the injection of bacterial cultures into the silkworm hemolymph, 15 strains killed all of the silkworms within 5 d. Additionally, bacterial strain 14 killed all of silkworms within 12 h with a median lethal dose of 4 × 10(4) colony-forming units/larva. This study provides useful information for healthcare safety in Chinese hospitals.

  13. The Chinese in Houston.

    ERIC Educational Resources Information Center

    Woodell, Thomas M.

    There are between seven and eight thousand Chinese Americans living in Houston, but there has never been a predominantly Chinese neighborhood in the city. This lack of geographical focus has prevented the development of easily identifiable aspects of ethnic concentration, such as a Chinese school or a Chinese business district. Apart from the…

  14. Molecular tracing of white muscardine in the silkworm, Bombyx mori (Linn.) II. Silkworm white muscardine is not caused by artificial release or natural epizootic of Beauveria bassiana in China.

    PubMed

    Chen, Xue; Huang, Cui; He, Lingmin; Zhang, Shengli; Li, Zengzhi

    2015-02-01

    The fungal pathogen Beauveria bassiana causes serious economic losses in sericulture. Its origin is usually attributed to the release of B. bassiana insecticides against pine caterpillars (Dendrolimus punctuatus). In the present study, 488 B. bassiana isolates obtained from silkworm (Bombyx mori) collected from 13 Chinese provinces, and 327 B. bassiana isolates obtained from D. punctatus collected from 9 provinces, were analyzed for population genetic structure using the ISSR technique based on genetic distance. A UPGMA dendrogram clustered them into three independent clades: two B. mori clades and one D. punctatus clade. A 3-D principal component analysis further divided them into two completely independent host groups, revealing high host-specificity. This suggested that white muscardine occurring in B. mori populations throughout southern China was not caused by any B. bassiana strain either naturally prevailing in D. punctatus populations or by any strain artificially released as a fungal insecticide against D. punctatus. We further investigated the genetic differentiation coefficient Gst and gene flow between B. mori-pathogenic and D. punctatus-pathogenic B. bassiana isolates from across China and from five provinces inhabited by both B. mori and D. punctatus. The Gst value across China was computed as 0.410, while the values of the five provinces ranged from 0.508 to 0.689; all above 0.25, which is the threshold for significant genetic differentiation. This suggests that B. bassiana strains isolated from the two different hosts maintained their respective heredity without a convergent homogenization trend, and reduces the possibility that the host range of the caterpillar isolates could expand and enhance their virulence in B. mori. These findings indicate that the use of B. bassiana does not threaten the safety of sericulture.

  15. Blocking binding of Bacillus thuringiensis Cry1Aa to Bombyx mori cadherin receptor results in only a minor reduction of toxicity

    PubMed Central

    You, Taek H; Lee, Mi K; Jenkins, Jeremy L; Alzate, Oscar; Dean, Donald H

    2008-01-01

    Background Bacillus thuringiensis Cry1Aa insecticidal protein is the most active known B. thuringiensis toxin against the forest insect pest Lymantria dispar (gypsy moth), unfortunately it is also highly toxic against the non-target insect Bombyx mori (silk worm). Results Surface exposed hydrophobic residues over domains II and III were targeted for site-directed mutagenesis. Substitution of a phenylalanine residue (F328) by alanine reduced binding to the Bombyx mori cadherin by 23-fold, reduced biological activity against B. mori by 4-fold, while retaining activity against Lymantria dispar. Conclusion The results identify a novel receptor-binding epitope and demonstrate that virtual elimination of binding to cadherin BR-175 does not completely remove toxicity in the case of B. mori. PMID:18218126

  16. Rhodiola rosea extends lifespan and improves stress tolerance in silkworm, Bombyx mori.

    PubMed

    Chen, Cong; Song, Jiangbo; Chen, Min; Li, Zhiquan; Tong, Xiaoling; Hu, Hai; Xiang, Zhonghuai; Lu, Cheng; Dai, Fangyin

    2016-04-01

    The root of Rhodiola rosea is widely used in Traditional Chinese Medicine. The extract from R. rosea is reported to extend the lifespan of yeast, nematode, and fruit fly. However, the molecular mechanism is not fully understood. Here, we tested whether R. rosea extends the lifespan of the silkworm. An aqueous extract of R. rosea significantly prolonged the lifespan of the silkworm, without affecting its daily food intake, body weight, or fecundity, suggesting that R. rosea did not exhibit obvious side effects. Rhodiola rosea extract also enhanced the stress resistance in the silkworm, against heat stress (37 °C) and starvation. The R. rosea extract increased the activity of the major antioxidant enzymes, glutathione S-transferase and catalase, and altered the content of glutathione and malondialdehyde. Rhodiola rosea increased the expression of BmFoxO, which is a downstream regulator of insulin/IGF-1 signaling (IIS) pathway in the silkworm. Our results showed that R. rosea extends lifespan, in which IIS pathway might be involved, and enhances stress resistance in the silkworm. Thus, the silkworm might be used as a novel animal model for lifespan study and efficacy evaluation of Traditional Chinese Medicines.

  17. Effect of Induced Oxidative Stress and Herbal Extracts on Acid Phosphatase Activity in Lysosomal and Microsomal Fractions of Midgut Tissue of the Silkworm, Bombyx mori

    PubMed Central

    Gaikwad, Y. B.; Gaikwad, S. M.; Bhawane, G. P.

    2010-01-01

    Lysosomal and microsomal acid phosphatase activity was estimated in midgut tissue of silkworm larvae, Bombyx mori L. (Lepidoptera: Bombycidae), after induced oxidative stress by D-galactose. The larvae were simultaneously were treated with ethanolic extracts of Bacopa monniera and Lactuca sativa to study their antioxidant properties. Lipid peroxidation and fluorescence was measured to analyze extent of oxidative stress. The ethanolic extract of Lactuca sativa was found to be more effective in protecting membranes against oxidative stress than Bacopa monniera. PMID:20874583

  18. [Effect of low-dose gamma-irradiation of silkworm eggs on the development of the mulberry silkworm (Bombyx mori L.)].

    PubMed

    Iusifov, N I; Kuzin, A M; Agaev, F A; Mozgovoĭ, E G

    1988-01-01

    Chronic gamma-irradiation of an egg at a dose-rate 100, 1000 and 4000 times as high as that of the natural radiation background significantly accelerates the development of Bombyx mori L. The caterpillar development may also be stimulated by a single exposure of the egg to 2 Gy radiation. The acceleration of the caterpillar growth promotes the increase in the cocoon weight and the raw-silk mass.

  19. The effect of the juvenile hormone analog, fenoxycarb, on ecdysone receptor B1 expression in the midgut of Bombyx mori during larval-pupal metamorphosis.

    PubMed

    Goncu, Ebru; Parlak, Osman

    2012-04-24

    The Bombyx mori (Lepidoptera: Bombycidae) midgut undergoes remodeling during the larval-pupal metamorphosis. All metamorphic events in insects are controlled by mainly two hormones: 20-hydroxyecdysone (20E) and juvenile hormone (JH). Fenoxycarb, O-ethyl N-(2-(4-phenoxyphenoxy)-ethyl) carbamate, has been shown to be one of the most potent juvenile hormone analogs against a variety of insect species. In this study, the effect of fenoxycarb on EcR-B1 protein expression in the midgut of Bombyx mori during the remodeling processwas investigated. Fenoxycarb was topically treated to the beginning of the fifth instar Bombyx larvae. Its application prolonged the last instar and prevented metamorphic events. Analyses were performed from day 6 of the fifth instar to 24 hr after pupation in controls and to day 14 of the fifth instar in the fenoxycarb treated group. According to our results, the presence of EcR-B1 in the midguts of the fenoxycarb treated group during the feeding period suggested that EcR-B1 was involved in the functioning of larval cells and during this period fenoxycarb did not affect EcR-B1 status. Immediately after termination of the feeding stage, the amount of EcR-B1 protein increased, which indicated that it may strengthen the ecdysone signal for commitment of remodeling process. In the fenoxycarb treated group, its upregulation was delayed, which may be related to the inhibition of ecdysone secretion from the prothoracic gland.

  20. Resistance to BmNPV via overexpression of an exogenous gene controlled by an inducible promoter and enhancer in transgenic silkworm, Bombyx mori.

    PubMed

    Jiang, Liang; Cheng, Tingcai; Zhao, Ping; Yang, Qiong; Wang, Genhong; Jin, Shengkai; Lin, Ping; Xiao, Yang; Xia, Qingyou

    2012-01-01

    The hycu-ep32 gene of Hyphantria cunea NPV can inhibit Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication in co-infected cells, but it is not known whether the overexpression of the hycu-ep32 gene has an antiviral effect in the silkworm, Bombyx mori. Thus, we constructed four transgenic vectors, which were under the control of the 39 K promoter of BmNPV (39 KP), Bombyx mori A4 promoter (A4P), hr3 enhancer of BmNPV combined with 39 KP, and hr3 combined with A4P. Transgenic lines were created via embryo microinjection using practical diapause silkworm. qPCR revealed that the expression level of hycu-ep32 could be induced effectively after BmNPV infection in transgenic lines where hycu-ep32 was controlled by hr3 combined with 39 KP (i.e., HEKG). After oral inoculation of BmNPV with 3 × 10(5) occlusion bodies per third instar, the mortality with HEKG-B was approximately 30% lower compared with the non-transgenic line. The economic characteristics of the transgenic lines remained unchanged. These results suggest that overexpression of an exogenous antiviral gene controlled by an inducible promoter and enhancer is a feasible method for breeding silkworms with a high antiviral capacity.

  1. Resistance to BmNPV via Overexpression of an Exogenous Gene Controlled by an Inducible Promoter and Enhancer in Transgenic Silkworm, Bombyx mori

    PubMed Central

    Jiang, Liang; Cheng, Tingcai; Zhao, Ping; Yang, Qiong; Wang, Genhong; Jin, Shengkai; Lin, Ping; Xiao, Yang; Xia, Qingyou

    2012-01-01

    The hycu-ep32 gene of Hyphantria cunea NPV can inhibit Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication in co-infected cells, but it is not known whether the overexpression of the hycu-ep32 gene has an antiviral effect in the silkworm, Bombyx mori. Thus, we constructed four transgenic vectors, which were under the control of the 39 K promoter of BmNPV (39 KP), Bombyx mori A4 promoter (A4P), hr3 enhancer of BmNPV combined with 39 KP, and hr3 combined with A4P. Transgenic lines were created via embryo microinjection using practical diapause silkworm. qPCR revealed that the expression level of hycu-ep32 could be induced effectively after BmNPV infection in transgenic lines where hycu-ep32 was controlled by hr3 combined with 39 KP (i.e., HEKG). After oral inoculation of BmNPV with 3 × 105 occlusion bodies per third instar, the mortality with HEKG-B was approximately 30% lower compared with the non-transgenic line. The economic characteristics of the transgenic lines remained unchanged. These results suggest that overexpression of an exogenous antiviral gene controlled by an inducible promoter and enhancer is a feasible method for breeding silkworms with a high antiviral capacity. PMID:22870254

  2. 20-Hydroxyecdysone stimulates nuclear accumulation of BmNep1, a nuclear ribosome biogenesis-related protein in the silkworm, Bombyx mori.

    PubMed

    Ji, M-M; Liu, A-Q; Sima, Y-H; Xu, S-Q

    2016-10-01

    The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth-instar ecdysis and larval-pupal metamorphosis. By immunoprecipitation with the anti-BmNep1 antibody and liquid chromatography-tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20-hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval-pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx. © 2016 The Royal Entomological Society.

  3. A homolog of the human Hermansky-Pudluck syndrome-5 (HPS5) gene is responsible for the oa larval translucent mutants in the silkworm, Bombyx mori.

    PubMed

    Fujii, T; Banno, Y; Abe, H; Katsuma, S; Shimada, T

    2012-12-01

    Normally, many granules containing uric acid accumulate in the larval integument of the silkworm, Bombyx mori. These uric acid granules cause the wild-type larval integument to be white or opaque, and the absence of these granules results in a translucent integument. Although about 30 B. mori loci governing larval translucency have been mapped, most have not been molecularly identified yet. Here, based on a structural analysis of a deletion of chromosome 14 that included the oa (aojyuku translucent) locus, we concluded that the BmHPS5 encoding a Bombyx homolog of the HPS5 subunit of biogenesis of lysosome-related organelles complex-2 is the candidate for the oa locus. Nucleotide sequence analyses of cDNAs and genomic DNAs in three mutant strains, each of which were homozygous for the respective allele of the oa locus (oa, oa ( 2 ), and oa ( v )), revealed that each mutant strain has a frame shift or a premature stop codon (caused by deletion or nonsense mutation, respectively) in the BmHPS5 gene. Our findings indicate that some genes that cause the translucent phenotype in Bombyx, some HPS-associated genes in humans, and some genes that cause mutant eye color phenotypes in Drosophila are homologous and participate in an evolutionarily conserved mechanism that leads to biogenesis of lysosome-related organelles.

  4. Happy (Chinese) New Year!

    ERIC Educational Resources Information Center

    Johnson, Georgia G.

    1979-01-01

    Suggestions are made for a classroom celebration of Chinese New Year, including discussion of the Chinese calendar and customs, a short list of appropriate children's stories, and food ideas, including a recipe for fortune cookies. (SJL)

  5. Molecular Cloning, Bioinformatic Analysis, and Expression of Bombyx mori Lebocin 5 Gene Related to Beauveria bassiana Infection

    PubMed Central

    Lü, Dingding; Hou, Chengxiang; Qin, Guangxing; Gao, Kun; Chen, Tian

    2017-01-01

    A full-length cDNA of lebocin 5 (BmLeb5) was first cloned from silkworm, Bombyx mori, by rapid amplification of cDNA ends. The BmLeb5 gene is 808 bp in length and the open reading frame encodes a 179-amino acid hydroxyproline-rich peptide. Bioinformatic analysis results showed that BmLeb5 owns an O-glycosylation site and four RXXR motifs as other lebocins. Sequence similarity and phylogenic analysis results indicated that lebocins form a multiple gene family in silkworm as cecropins. Quantitative real-time PCR analysis revealed that BmLeb5 was highest expressed in the fat body. In the silkworm larvae infected by Beauveria bassiana, the expression level of BmLeb5 was upregulated in the fat body and hemolymph which are the most important immune tissues in silkworm. The recombinant protein of BmLeb5 was for the first time successfully expressed with prokaryotic expression system and purified. There are no reports so far that the expression of lebocins could be induced by entomopathogenic fungus. Our study suggested that BmLeb5 might play an important role in the immune response of silkworm to defend B. bassiana infection. The results also provided helpful information for further studying the lebocin family functioned in antifungal immune response in the silkworm. PMID:28194425

  6. Recent transposition of yabusame, a novel piggyBac-like transposable element in the genome of the silkworm, Bombyx mori.

    PubMed

    Daimon, Takaaki; Mitsuhiro, Masao; Katsuma, Susumu; Abe, Hiroaki; Mita, Kazuei; Shimada, Toru

    2010-08-01

    On the W chromosome of the silkworm, Bombyx mori, we found a novel piggyBac-like DNA transposon that potentially encodes an intact transposase (610 amino acid residues), which is flanked by 16-bp perfect inverted terminal repeats and a duplicated TTAA target site. Interestingly, we also identified another intact copy of this transposon on an autosome (chromosome 21), which showed 99.6% identity in the DNA sequence of the transposase (99.3% amino acid identity). These features raised the possibility that this novel piggyBac-like DNA transposon, designated as yabusame, may retain transposition activity. Here we report the identification and characterization of yabusame transposons from the silkworm. We cloned the full length of the yabusame transposon on the W chromosome (yabusame-W) and its autosomal copy (yabusame-1). Southern blot analysis showed that there are interstrain polymorphisms in yabusame elements for their insertion sites and copy number. We also found strong evidence for the recent transposition of yabusame elements in the silkworm genome. Although our in vitro excision assays suggested that the transposition activity of yabusame-1 and yabusame-W has been lost almost entirely, our data will lead to a greater understanding of the characteristics of piggyBac superfamily elements.

  7. Molecular characterization of DnaJ 5 homologs in silkworm Bombyx mori and its expression during egg diapause.

    PubMed

    Sirigineedi, Sasibhushan; Vijayagowri, Esvaran; Murthy, Geetha N; Rao, Guruprasada; Ponnuvel, Kangayam M

    2014-12-01

    A comparison of the cDNA sequences (1 056 bp) of Bombyx mori DnaJ 5 homolog with B. mori genome revealed that unlike in other Hsps, it has an intron of 234 bp. The DnaJ 5 homolog contains 351 amino acids, of which 70 contain the conserved DnaJ domain at the N-terminal end. This homolog of B. mori has all desirable functional domains similar to other insects, and the 13 different DnaJ homologs identified in B. mori genome were distributed on different chromosomes. The expressed sequence tag database analysis of Hsp40 gene expression revealed higher expression in wing disc followed by diapause-induced eggs. Microarray analysis revealed higher expression of DnaJ 5 homolog at 18th h after oviposition in diapause-induced eggs. Further validation of DnaJ 5 expression through qPCR in diapause-induced and nondiapause eggs at different time intervals revealed higher expression in diapause eggs at 18 and 24 h after oviposition, which coincided with the expression of Hsp70 as the Hsp 40 is its co-chaperone. This study thus provides an outline of the genome organization of Hsp40 gene, and its role in egg diapause induction in B. mori.

  8. Effects of alkyl polyglycoside (APG) on Bombyx mori silk degumming and the mechanical properties of silk fibroin fibre.

    PubMed

    Wang, Fei; Zhang, Yu-Qing

    2017-05-01

    Alkyl polyglycoside (APG), a nonionic surfactant, is often considered to be a green surfactant and is synthesized using glucose and long chain fatty alcohols. It is used as a degumming agent of Bombyx mori silk fibre in this study for the first time. We studied APG systematically in comparison to the traditional degumming methods, such as aqueous solutions of sodium carbonate (Na2CO3) and neutral soap (NS). After repeatedly boiling silk fibres in an aqueous solution of 0.25% APG three times for 30min and using a bath ratio of 1:90-120 (g/mL), sericin was completely removed from the fibre. SDS-PAGE showed that the degumming in APG did not induce an evident breakage of the silk fibroin peptide chains, including the light chain and P25 protein. The tensile properties, thermal analysis, and scanning electron microscopic (SEM) observation of the degummed fibroin fibre all show that APG is a degumming agent similar to NS and far superior to Na2CO3. These results indicate that APG is an environment-friendly silk degumming/refining agent in the silk textile industry and in the manufacture of silk floss quilts. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Isolation, purification, and identification of an important pigment, sepiapterin, from integument of the lemon mutant of the silkworm, Bombyx mori.

    PubMed

    Gao, Junshan; Wang, Jing; Wang, Wenjing; Liu, Chaoliang; Meng, Yan

    2013-01-01

    Sepiapterin is the precursor of tetrahydrobiopterin, an important coenzyme of aromatic amino acid hydroxylases, the lack of which leads to a variety of physiological metabolic diseases or neurological syndromes in humans. Sepiapterin is a main pigment component in the integument of the lemon mutant of the silkworm, Bombyx mori (L.) (Lepidoptera: Bombycidae), and is present there in extremely high content, so lemon is a valuable genetic resource to extract sepiapterin. In this study, an effective experimental system was set up for isolation and purification of sepiapterin from lemon silkworms by optimizing homogenization solvent, elution buffer, and separation chromatographic column. The results showed that ethanol was the most suitable solvent to homogenize the integument, with a concentration of 50% and solid:liquid ratio of 1:20 (g/mL). Sepiapterin was purified successively by column chromatography of cellulose Ecteola, sephadex G-25-150, and cellulose phosphate, and was identified by ultraviolet-visible absorption spectrometry. A stable and accurate high performance liquid chromatography method was constructed to identify sepiapterin and conduct qualitative and quantitative analyses. Sepiapterin of high purity was achieved, and the harvest reached about 40 ug/g of integument in the experiments. This work helps to obtaining natural sepiapterin in large amounts in order to use the lemon B. mori mutant to produce BH4 in vitro.

  10. Analyis of structure/property relationships in silkworm (Bombyx mori) and spider dragline (Nephila edulis) silks using Raman spectroscopy.

    PubMed

    Sirichaisit, Jutarat; Brookes, Victoria L; Young, Robert J; Vollrath, Fritz

    2003-01-01

    The molecular deformation of both silkworm (Bombyx mori) and spider dragline (Nephila edulis) silks has been studied using a combination of mechanical deformation and Raman spectroscopy. The stress/strain curves for both kinds of silk showed elastic behavior followed by plastic deformation. It was found that both materials have well-defined Raman spectra and that some of the bands in the spectra shift to lower frequency under the action of tensile stress or strain. The band shift was linearly dependent upon stress for both types of silk fiber. This observation provides a unique insight into the effect of tensile deformation upon molecular structure and the relationship between structure and mechanical properties. Two similar bands in the Raman spectra of both types of silk in the region of 1000-1300 cm(-1) had significant identical rates of Raman band shift of about 7 cm(-1)/GPa and 14 cm(-1)/GPa demonstrating the similarity between the silk fibers from two different animals.

  11. Glycerol-3-phosphate O-acyltransferase is required for PBAN-induced sex pheromone biosynthesis in Bombyx mori.

    PubMed

    Du, Mengfang; Liu, Xiaoguang; Liu, Xiaoming; Yin, Xinming; Han, Shuangyin; Song, Qisheng; An, Shiheng

    2015-01-29

    Female moths employ their own pheromone blends as a communicational medium in mating behavior. The biosynthesis and release of sex pheromone in female moths are regulated by pheromone biosynthesis activating neuropeptide (PBAN) and the corresponding action of PBAN has been well elucidated in Bombyx mori. However, very little is known about the molecular mechanism regarding the biosynthesis of sex pheromone precursor. In this study, quantitative proteomics was utilized to comprehensively elucidate the expression dynamics of pheromone glands (PGs) during development. Proteomic analysis revealed a serial of differentially expressed sex pheromone biosynthesis-associated proteins at the different time points of B. mori development. Most interestingly B. mori glycerol-3-phosphate O-acyltransferase (BmGPAT) was found to be expressed during the key periods of sex pheromone biosynthesis. RNAi knockdown of BmGPAT confirmed the important function of this protein in the biosynthesis of sex pheromone precursor, triacylglcerol (TAG), and subsequently PBAN-induced production of sex pheromone, bombykol. Behavioral analysis showed that RNAi knockdown of GPAT significantly impaired the ability of females to attract males. Our findings indicate that GPAT acts to regulate the biosynthesis of sex pheromone precursor, TAG, thus influencing PBAN-induced sex pheromone production and subsequent mating behavior.

  12. The origin and evolution of six miniature inverted-repeat transposable elements in Bombyx mori and Rhodnius prolixus.

    PubMed

    Zhang, Hua-Hao; Xu, Hong-En; Shen, Yi-Hong; Han, Min-Jin; Zhang, Ze

    2013-01-01

    Miniature inverted-repeat transposable elements (MITEs) are a specific group of nonautonomous DNA transposons, and they are distributed in a wide range of hosts. However, the origin and evolutionary history of MITEs in eukaryotic genomes remain unclear. In this study, six MITEs were identified in the silkworm (Bombyx mori). Five elements are grouped into four known superfamilies of DNA transposons, and one represents a novel class of MITEs. Unexpectedly, six similar MITEs are also present in the triatomine bug (Rhodnius prolixus) that diverged from the common ancestor with the silkworm about 370 Ma. However, they show different lengths in two species, suggesting that they are different derivatives of progenitor transposons. Three direct progenitor transposons (Sola1, hobo/Ac/Tam [hAT], and Ginger2) are also identified in some other organisms, and several lines of evidence suggested that these autonomous elements might have been independently and horizontally transferred into their hosts. Furthermore, it is speculated that the twisted-wing parasites may be the candidate vectors for these horizontal transfers. The data presented in this study provide some new insights into the origin and evolutionary history of MITEs in the silkworm and triatomine bug.

  13. Characterization of sericin powder prepared from citric acid-degraded sericin polypeptides of the silkworm, Bombyx Mori.

    PubMed

    Kurioka, Akira; Kurioka, Fujie; Yamazaki, Masayoshi

    2004-04-01

    Acid-degraded sericin powder (AC-SP) was prepared from aqueous solution containing citric acid-degraded sericin polypeptides of Bombyx mori. The morphological and biochemical properties of AC-SP were compared with those of alkali-degraded sericin powder (AL-SP) and hot-water degraded sericin powder (HW-SP). Based on an SEM analysis, AC-SP showed a thin film structure of 10-100 microm with good dispersity while AL-SP and HW-SP had a much larger thin film structure (<500 microm). The extract of AC-SP showed stronger trypsin inhibitor activity due to cocoon shell trypsin inhibitor (CSTI-IV) than that of HW-SP. The extract of AL-SP showed no CSTI-IV activity. It was found that AC-SP was a trypsin inhibitor complex powder and that the release of CSTI-IV from AC-SP depended on pH and ion strength. Similar powder materials were obtained when such organic acids as tartaric acid and succinic acid were used. These results suggest that the acid-degraded sericin polypeptides work as a protein matrix to which CSTI-IV may bind ionically.

  14. The fat body cell-free system for tissue-specific transcription of plasma protein gene of Bombyx mori.

    PubMed Central

    Mine, E; Sakurai, H; Izumi, S; Tomino, S

    1995-01-01

    A nuclear extract was prepared for the larval fat body of the silkworm, Bombyx mori, and a homologous in vitro system was developed for the transcription of major plasma protein gene of B.mori. The gene for SP1, a storage protein of B.mori, and adenovirus 2 major late (AdML) gene were faithfully transcribed under relatively high template concentrations in the nuclear extract prepared from the fat body of female fifth instar larvae. Complete inhibition of gene transcription by a low concentration of alpha-amanitin indicated that the reaction is catalyzed by RNA polymerase II. At low template concentration (0.6 nM) the fat body nuclear extract transcribed the homologous SP1 gene with high efficiency, while AdML gene and larval cuticle protein gene were only barely transcribed in the same extract. The SP1 gene deleted upstream of the TATA box sequence showed little effect on transcription, whereas mutations that destroy TATA sequence totally abolished the gene transcription. These results suggested that the core promoter region of SP1 gene spanning between positions -44 and +16 is essential for the fat body specific transcription in vitro. Images PMID:7651825

  15. The fat body cell-free system for tissue-specific transcription of plasma protein gene of Bombyx mori.

    PubMed

    Mine, E; Sakurai, H; Izumi, S; Tomino, S

    1995-07-25

    A nuclear extract was prepared for the larval fat body of the silkworm, Bombyx mori, and a homologous in vitro system was developed for the transcription of major plasma protein gene of B.mori. The gene for SP1, a storage protein of B.mori, and adenovirus 2 major late (AdML) gene were faithfully transcribed under relatively high template concentrations in the nuclear extract prepared from the fat body of female fifth instar larvae. Complete inhibition of gene transcription by a low concentration of alpha-amanitin indicated that the reaction is catalyzed by RNA polymerase II. At low template concentration (0.6 nM) the fat body nuclear extract transcribed the homologous SP1 gene with high efficiency, while AdML gene and larval cuticle protein gene were only barely transcribed in the same extract. The SP1 gene deleted upstream of the TATA box sequence showed little effect on transcription, whereas mutations that destroy TATA sequence totally abolished the gene transcription. These results suggested that the core promoter region of SP1 gene spanning between positions -44 and +16 is essential for the fat body specific transcription in vitro.

  16. Free radical scavenging and tyrosinase inhibition activity of oils and sericin extracted from Thai native silkworms (Bombyx mori).

    PubMed

    Manosroi, Aranya; Boonpisuttinant, Korawinwich; Winitchai, Supanida; Manosroi, Worapaka; Manosroi, Jiradej

    2010-08-01

    Oils and sericin were extracted from pupae and silk cocoons, respectively, of the five Thai native silkworms (Bombyx mori, Linnaeus (Bombycidae)), namely, Keawsakol, Nangnoi, Somrong, Nangleung, and Noneruesee, which are variations of the same species. The oils were extracted by a hot process using Soxhlet apparatus and a cold process using petroleum ether, while sericin was extracted by basic hydrolysis and autoclaving. Sericin from the five Thai native silkworms showed free radical scavenging activity lower than the standard antioxidants (vitamin C, vitamin E, and BHT) by about 20-100-fold, but all oils gave higher activity than that of the standard linoleic acid by 11-22-fold. Oil extracted from Noneruesee by the cold process gave the highest DPPH scavenging activity, compared with other oil samples. All sericin samples showed tyrosinase inhibition activity with IC(50) values in the range of 1.2-18.76 mg/mL, but only oils from Noneruesee extracted by the hot process, and Nangleung, Somrong, and Noneruesee extracted by the cold process, showed this activity. Oil extracted by the hot process and sericin by basic hydrolysis from Noneruesee gave the highest tyrosinase inhibition activity, but lower than that of the standards vitamin C and kojic acid by 20-49 and 3-8 times, respectively. This study has suggested that sericin and oil from Noneruesee extracted by basic hydrolysis and the cold process, which gave the highest tyrosinase inhibition and free radical scavenging activity, respectively, can be applied in antiaging and whitening cosmetic products.

  17. IR study on the binding mode of metal cations to chemically modified Bombyx mori and Tussah silk fibres

    NASA Astrophysics Data System (ADS)

    Taddei, Paola; Monti, Patrizia; Freddi, Giuliano; Arai, Takayuki; Tsukada, Masuhiro

    2003-06-01

    Bombyx mori ( B. mori) and Tussah ( Antheraea pernyi) silk fibres were modified by treatment with tannic acid (TA) or ethylenediaminetetraacetic (EDTA) dianhydride, subsequently treated with Cu 2+ and Co 2+ solutions, at alkaline pH, and analysed by attenuated total reflectance/infrared spectroscopy to evaluate the changes induced in their structure by metal binding. The spectral changes were correlated to metal binding results obtained by inductive coupled plasma-atomic emission spectrometry. Upon Co 2+ complexation, the spectra of all the B. mori and Tussah silk samples showed a decrease in intensity of the Amide I band with a trend related to the metal uptake. The most relevant changes in the whole spectra were observed for the EDTA- and TA-modified samples in the case of B. mori and Tussah silks, respectively. Upon Cu 2+ complexation, the decrease in intensity of the Amide I band follows the trend of the metal uptake only in the case of Tussah silk. Moreover, the most relevant changes with respect to the untreated sample were observed for the TA-modified B. mori silk sample. The spectral changes were explained by considering the different affinities of the fibres for the modifying reagent, the amount of the metal bound and the relative stability of the complexes formed.

  18. Morphological characterization and molecular mapping of an irradiation-induced Speckled mutant in the silkworm, Bombyx mori.

    PubMed

    Tan, D; Tong, X-L; Hu, H; Wu, S-Y; Li, C-L; Xiong, G; Xiang, Z-H; Dai, F-Y; Lu, C

    2016-04-01

    Speckled (Spc), an X-ray-induced lethal mutant of Bombyx mori, exhibits a mosaic dark-brown-spotted larval epidermis in both sexes and egg-laying problems only in females. Here, we report the morphological characterization and molecular mapping of the Spc mutant. Morphological investigations revealed that the epidermal ultrastructure of the small, dark-brown spots was more dense than that of the white regions in both Spc/+ mutants and wild type, and that the lethality of the Spc/Spc mutants occurred during early embryogenesis. Furthermore, the ovarioles and ovipositor were disconnected in approximately 85.5% of Spc/+ females, a further 2.5% had a connection between the ovarioles and ovipositor that was too narrow to lay eggs. The remaining females showed a normal connection similar to that of the wild type. We successfully narrowed down the location of the Spc mutation to a region on chromosome 4 that was ∼1041 kb long. Gene-prediction analysis identified 25 candidate genes in this region. Chromosome structure analysis indicated that a ∼305 kb deletion was included in the mapping region. Temporal and spatial reverse transcription PCR (RT-PCR) analysis showed that several genes in the mapped region are associated with the Spc mutant. Although the genes responsible for the Spc mutation were not definitively identified, our results further the current understanding of the complex mechanism underlying the multiple morphological defects in Spc mutants. © 2015 The Royal Entomological Society.

  19. A composite method for mapping quantitative trait loci without interference of female achiasmatic and gender effects in silkworm, Bombyx mori.

    PubMed

    Li, C; Zuo, W; Tong, X; Hu, H; Qiao, L; Song, J; Xiong, G; Gao, R; Dai, F; Lu, C

    2015-08-01

    The silkworm, Bombyx mori, is an economically important insect that was domesticated more than 5000 years ago. Its major economic traits focused on by breeders are quantitative traits, and an accurate and efficient QTL mapping method is necessary to explore their genetic architecture. However, current widely used QTL mapping models are not well suited for silkworm because they ignore female achiasmate and gender effects. In this study, we propose a composite method combining rational population selection and special mapping methods to map QTL in silkworm. By determining QTL for cocoon shell weight (CSW), we demonstrated the effectiveness of this method. In the CSW mapping process, only 56 markers were used and five loci or chromosomes were detected, more than in previous studies. Additionally, loci on chromosomes 1 and 11 dominated and accounted for 35.10% and 15.03% of the phenotypic variance respectively. Unlike previous studies, epistasis was detected between loci on chromosomes 11 and 22. These mapping results demonstrate the power and convenience of this method for QTL mapping in silkworm, and this method may inspire the development of similar approaches for other species with special genetic characteristics. © 2015 Stichting International Foundation for Animal Genetics.

  20. Characterization of a protein tyrosine phosphatase as a host factor promoting baculovirus replication in silkworm, Bombyx mori.

    PubMed

    Wang, Fei; Xue, Renju; Li, Xianyang; Hu, Cuimei; Xia, Qingyou

    2016-04-01

    The relevance of protein tyrosine phosphatase (PTP) to host-pathogen interaction is highlighted in mammalian studies, whereas less is known in insects. Here we presented the categorization of the PTP complement of silkworm and characterized their homologous relationship with human and fruit fly PTPs. Among the 36 PTP genes, ptp-h, which was proposed to be the origin of baculovirus ptp belongs to atypical VH1-like dual-specific PTP subset and encodes a catalytic active protein. The maximum expression level of Bmptp-h was at 5th instar and in fat body. Bombyx mori nucleopolyhedrovirus (BmNPV) infection potently induced its expression in silkworm larvae and in BmE cells. Knock-down of Bmptp-h by RNA interference significantly inhibited viral replication, and over-expression enhanced viral replication as determined by viral DNA abundance and BmNPV-GFP positive cells. These results suggest that BmPTP-h might be one of the host factors that is beneficial to baculovirus infection by promoting viral replication. Copyright © 2015. Published by Elsevier Ltd.

  1. Molecular cloning, characterization and expression analysis of cathepsin O in silkworm Bombyx mori related to bacterial response.

    PubMed

    Zhang, Kui; Su, Jingjing; Chen, Siyuan; Yu, Shuang; Tan, Juan; Xu, Man; Liang, Hanghua; Zhao, Yuzu; Chao, Huijuan; Yang, Liqun; Cui, Hongjuan

    2015-08-01

    Cathepsins are the main members of the cysteine family and play important roles in immune response in vertebrates. The Cathepsin O of Bombyx mori (BmCathepsin O) was cloned from the hemocytes by the rapid amplification of cDNA ends (RACE). The genomic DNA was 6131bp long with a total of six exons and five introns. Its pre-mRNA was spliced to generate two spliceosomes. By comparisons with other reported cathepsins O, it was concluded that the identity between them ranged from 29 to 39%. Expression analysis indicated that BmCathepsin O was specific-expressed in hemocytes, and highly expressed at the 4th molting and metamorphosis stages. Immunofluorescence assay and qRT-PCR showed that BmCathepsin O was expressed in granulocytes and plasmatocytes. Interestingly, BmCathepsin O was significantly up-regulated after stimulated by 20-hydroxyecdysone (20-E) in vivo, which suggested that BmCathepsin O may be regulated by 20E. Moreover, activation of BmCathepsin O was also observed in hemocytes challenged by Escherichia coli, indicating its potential involvement in the innate immune system of silkworm, B. mori. In summary, our studies provide a new insight into the functional features of Cathepsin O. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Abortive replication of Bombyx mori nucleopolyhedrovirus in Sf9 and High Five cells: Defective nuclear transport of the virions

    SciTech Connect

    Katou, Yasuhiro; Ikeda, Motoko; Kobayashi, Michihiro . E-mail: michihir@agr.nagoya-u.ac.jp

    2006-04-10

    Despite close genetic relationship, Bombyx mori nucleopolyhedrovirus (BmNPV) and Autographa californica multicapsid NPV (AcMNPV) display a distinct host range property. Here, BmNPV replication was examined in Sf9 and High Five cells that were nonproductive for BmNPV infection but supported high titers of AcMNPV replication. Recombinant BmNPV, vBm/gfp/lac, containing bm-ie1 promoter-driven egfp showed that few Sf9 and High Five cells infected with vBm/gfp/lac expressed EGFP, while large proportion of EGFP-expressing cells was observed when transfected with vBm/gfp/lac DNA. Immunocytochemical analysis showed that BmNPV was not imported into the nucleus of these two cell lines, while recombinant BmNPV, vBm{delta}64/ac-gp64 possessing AcMNPV gp64 was imported into the nucleus, yielding progeny virions in High Five cells, but not Sf9 cells. These results indicate that the defective nuclear import of infected virions due to insufficient BmNPV GP64 function is involved in the restricted BmNPV replication in Sf9 and High Five cells.

  3. Analysis of differentially expressed genes between fluoride-sensitive and fluoride-endurable individuals in midgut of silkworm, Bombyx mori.

    PubMed

    Qian, Heying; Li, Gang; He, Qingling; Zhang, Huaguang; Xu, Anying

    2016-08-15

    Fluoride tolerance is an economically important trait of silkworm. Near-isogenic lines (NILs) of the dominant endurance to fluoride (Def) gene in Bombyx mori has been constructed before. Here, we analyzed the gene expression profiles of midgut of fluoride-sensitive and fluoride-endurable individuals of Def NILs by using high-throughput Illumina sequencing technology and bioinformatics tools, and identified differentially expressed genes between these individuals. A total of 3,612,399 and 3,567,631 clean tags for the libraries of fluoride-endurable and fluoride-sensitive individuals were obtained, which corresponded to 32,933 and 43,976 distinct clean tags, respectively. Analysis of differentially expressed genes indicates that 241 genes are differentially expressed between the two libraries. Among the 241 genes, 30 are up-regulated and 211 are down-regulated in fluoride-endurable individuals. Pathway enrichment analysis demonstrates that genes related to ribosomes, pancreatic secretion, steroid biosynthesis, glutathione metabolism, steroid biosynthesis, and glycerolipid metabolism are down-regulated in fluoride-endurable individuals. qRT-PCR was conducted to confirm the results of the DGE. The present study analyzed differential expression of related genes and tried to find out whether the crucial genes were related to fluoride detoxification which might elucidate fluoride effect and provide a new way in the fluorosis research. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. BmICE-2 is a novel pro-apoptotic caspase involved in apoptosis in the silkworm, Bombyx mori.

    PubMed

    Yi, Hua-Shan; Pan, Cai-Xia; Pan, Chun; Song, Juan; Hu, Yan-Fen; Wang, La; Pan, Min-Hui; Lu, Cheng

    2014-02-28

    In this study we identified a potential pro-apoptotic caspase gene, Bombyx mori(B. mori)ICE-2 (BmICE-2) which encoded a polypeptide of 284 amino acid residues, including a (169)QACRG(173) sequence which surrounded the catalytic site and contained a p20 and a p10 domain. BmICE-2 expressed in Escherichia coli (E. coli) exhibited high proteolytic activity for the synthetic human initiator caspase-9 substrates Ac-LEHD-pNA, but little activity towards the effector caspase-3 substrates Ac-DEVD-pNA. When BmICE-2 was transiently expressed in BmN-SWU1 silkworm B. mori cells, we found that the high proteolytic activity for Ac-LEHD-pNA triggered caspase-3-like protease activity resulting in spontaneous cleavage and apoptosis in these cells. This effect was not replicated in Spodoptera frugiperda 9 cells. In addition, spontaneous cleavage of endogenous BmICE-2 in BmN-SWU1 cells could be induced by actinomycin D. These results suggest that BmICE-2 may be a novel pro-apoptotic gene with caspase-9 activity which is involved apoptotic processes in BmN-SWU1 silkworm B. mori cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Genome-Wide Identification, Characterization and Expression Analysis of the Solute Carrier 6 Gene Family in Silkworm (Bombyx mori)

    PubMed Central

    Tang, Xin; Liu, Huawei; Chen, Quanmei; Wang, Xin; Xiong, Ying; Zhao, Ping

    2016-01-01

    The solute carrier 6 (SLC6) gene family, initially known as the neurotransmitter transporters, plays vital roles in the regulation of neurotransmitter signaling, nutrient absorption and motor behavior. In this study, a total of 16 candidate genes were identified as SLC6 family gene homologs in the silkworm (Bombyx mori) genome. Spatio-temporal expression patterns of silkworm SLC6 gene transcripts indicated that these genes were highly and specifically expressed in midgut, brain and gonads; moreover, these genes were expressed primarily at the feeding stage or adult stage. Levels of expression for most midgut-specific and midgut-enriched gene transcripts were down-regulated after starvation but up-regulated after re-feeding. In addition, we observed that expression levels of these genes except for BmSLC6-15 and BmGT1 were markedly up-regulated by a juvenile hormone analog. Moreover, brain-enriched genes showed differential expression patterns during wandering and mating processes, suggesting that these genes may be involved in modulating wandering and mating behaviors. Our results improve our understanding of the expression patterns and potential physiological functions of the SLC6 gene family, and provide valuable information for the comprehensive functional analysis of the SLC6 gene family. PMID:27706106

  6. An efficient strategy for producing a stable, replaceable, highly efficient transgene expression system in silkworm, Bombyx mori.

    PubMed

    Long, Dingpei; Lu, Weijian; Zhang, Yuli; Bi, Lihui; Xiang, Zhonghuai; Zhao, Aichun

    2015-03-05

    We developed an efficient strategy that combines a method for the post-integration elimination of all transposon sequences, a site-specific recombination system, and an optimized fibroin H-chain expression system to produce a stable, replaceable, highly efficient transgene expression system in the silkworm (Bombyx mori) that overcomes the disadvantages of random insertion and post-integration instability of transposons. Here, we generated four different transgenic silkworm strains, and of one the transgenic strains, designated TS1-RgG2, with up to 16% (w/w) of the target protein in the cocoons, was selected. The subsequent elimination of all the transposon sequences from TS1-RgG2 was completed by the heat-shock-induced expression of the transposase in vivo. The resulting transgenic silkworm strain was designated TS3-g2 and contained only the attP-flanked optimized fibroin H-chain expression cassette in its genome. A phiC31/att-system-based recombinase-mediated cassette exchange (RMCE) method could be used to integrate other genes of interest into the same genome locus between the attP sites in TS3-g2. Controlling for position effects with phiC31-mediated RMCE will also allow the optimization of exogenous protein expression and fine gene function analyses in the silkworm. The strategy developed here is also applicable to other lepidopteran insects, to improve the ecological safety of transgenic strains in biocontrol programs.

  7. Translationally Controlled Tumor Protein, a Dual Functional Protein Involved in the Immune Response of the Silkworm, Bombyx mori

    PubMed Central

    Hua, Xiaoting; Song, Liang; Xia, Qingyou

    2013-01-01

    Insect gut immunity is the first line of defense against oral infection. Although a few immune-related molecules in insect intestine has been identified by genomics or proteomics approach with comparison to well-studied tissues, such as hemolymph or fat body, our knowledge about the molecular mechanism underlying the gut immunity which would involve a variety of unidentified molecules is still limited. To uncover additional molecules that might take part in pathogen recognition, signal transduction or immune regulation in insect intestine, a T7 phage display cDNA library of the silkworm midgut is constructed. By use of different ligands for biopanning, Translationally Controlled Tumor Protein (TCTP) has been selected. BmTCTP is produced in intestinal epithelial cells and released into the gut lumen. The protein level of BmTCTP increases at the early time points during oral microbial infection and declines afterwards. In vitro binding assay confirms its activity as a multi-ligand binding molecule and it can further function as an opsonin that promotes the phagocytosis of microorganisms. Moreover, it can induce the production of anti-microbial peptide via a signaling pathway in which ERK is required and a dynamic tyrosine phosphorylation of certain cytoplasmic membrane protein. Taken together, our results characterize BmTCTP as a dual-functional protein involved in both the cellular and the humoral immune response of the silkworm, Bombyx mori. PMID:23894441

  8. BmRobo1a and BmRobo1b control axon repulsion in the silkworm Bombyx mori.

    PubMed

    Li, Xiao-Tong; Yu, Qi; Zhou, Qi-Sheng; Zhao, Xiao; Liu, Zhao-Yang; Cui, Wei-Zheng; Liu, Qing-Xin

    2016-02-15

    The development of the nervous system is based on the growth and connection of axons, and axon guidance molecules are the dominant regulators during this course. Robo, as the receptor of axon guidance molecule Slit, plays a key role as a conserved repellent cue for axon guidance during the development of the central nervous system. However, the function of Robo in the silkworm Bombyx mori is unknown. In this study, we cloned two novel robo genes in B. mori (Bmrobo1a and Bmrobo1b). BmRobo1a and BmRobo1b lack an Ig and a FNIII domain in the extracellular region and the CC0 and CC2 motifs in the intracellular region. BmRobo1a and BmRobo1b were colocalized with BmSlit in the neuropil. Knock-down of Bmrobo1a and Bmrobo1b by RNA interference (RNAi) resulted in abnormal development of axons. Our results suggest that BmRobo1a and BmRobo1b have repulsive function in axon guidance, even though their structures are different from Robo1 of other species. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Genome-Wide Identification, Characterization and Expression Analysis of the Solute Carrier 6 Gene Family in Silkworm (Bombyx mori).

    PubMed

    Tang, Xin; Liu, Huawei; Chen, Quanmei; Wang, Xin; Xiong, Ying; Zhao, Ping

    2016-10-03

    The solute carrier 6 (SLC6) gene family, initially known as the neurotransmitter transporters, plays vital roles in the regulation of neurotransmitter signaling, nutrient absorption and motor behavior. In this study, a total of 16 candidate genes were identified as SLC6 family gene homologs in the silkworm (Bombyx mori) genome. Spatio-temporal expression patterns of silkworm SLC6 gene transcripts indicated that these genes were highly and specifically expressed in midgut, brain and gonads; moreover, these genes were expressed primarily at the feeding stage or adult stage. Levels of expression for most midgut-specific and midgut-enriched gene transcripts were down-regulated after starvation but up-regulated after re-feeding. In addition, we observed that expression levels of these genes except for BmSLC6-15 and BmGT1 were markedly up-regulated by a juvenile hormone analog. Moreover, brain-enriched genes showed differential expression patterns during wandering and mating processes, suggesting that these genes may be involved in modulating wandering and mating behaviors. Our results improve our understanding of the expression patterns and potential physiological functions of the SLC6 gene family, and provide valuable information for the comprehensive functional analysis of the SLC6 gene family.

  10. Crystal structure of Bombyx mori arylphorins reveals a 3:3 heterohexamer with multiple papain cleavage sites

    PubMed Central

    Hou, Yong; Li, Jianwei; Li, Yi; Dong, Zhaoming; Xia, Qingyou; Yuan, Y Adam

    2014-01-01

    In holometabolous insects, the accumulation and utilization of storage proteins (SPs), including arylphorins and methionine-rich proteins, are critical for the insect metamorphosis. SPs function as amino acids reserves, which are synthesized in fat body, secreted into the larval hemolymph and taken up by fat body shortly before pupation. However, the detailed molecular mechanisms of digestion and utilization of SPs during development are largely unknown. Here, we report the crystal structure of Bombyx mori arylphorins at 2.8 Å, which displays a heterohexameric structural arrangement formed by trimerization of dimers comprising two structural similar arylphorins. Our limited proteolysis assay and microarray data strongly suggest that papain-like proteases are the major players for B. mori arylphorins digestion in vitro and in vivo. Consistent with the biochemical data, dozens of papain cleavage sites are mapped on the surface of the heterohexameric structure of B. mori arylphorins. Hence, our results provide the insightful information to understand the metamorphosis of holometabolous insects at molecular level. PMID:24639361

  11. BmDredd is an initiator caspase and participates in Emodin-induced apoptosis in the silkworm, Bombyx mori.

    PubMed

    Wang, La; Song, Juan; Bao, Xi-Yan; Chen, Peng; Yi, Hua-Shan; Pan, Min-Hui; Lu, Cheng

    2016-10-15

    The identification and analysis of the caspases is essential to research into apoptosis in lepidoptera insects. The domesticated silkworm, Bombyx mori, is the model system for lepidopterans. In this study, we cloned and characterized a B. mori Dredd gene, BmDredd, the proposed insect homologue of human caspase-8, which encoded a polypeptide of 543 amino acids. BmDredd possesses a long N-terminal prodomain, a p20 domain, and a p10 domain. When transiently expressed in Escherichia coli cells, BmDredd underwent spontaneous cleavage and exhibited high proteolytic activity for caspase-8 substrate but relatively low for caspase-3 or -9 substrate. In addition, BmDredd induced apoptosis when transiently expressed in BmN-SWU1 cells, an ovarian cell line of B. mori. Moreover, after the treatment of Emodin, a novel apoptosis inducer, endogenous BmDredd expression level, the caspase-8 activity and the apoptotic rate increased notably in BmN-SWU1 cells. When BmDredd was subjected to interference in BmN-SWU1 cells and Emodin treatment, BmDredd expression levels decreased and the apoptotic rate also decreased significantly. These results suggest BmDredd is the homologue of human caspase-8 and plays a role in Emodin-induced apoptosis in BmN-SWU1 cells of B. mori. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. The effects of Bombyx mori silk strain and extraction time on the molecular and biological characteristics of sericin.

    PubMed

    Siritientong, Tippawan; Bonani, Walter; Motta, Antonella; Migliaresi, Claudio; Aramwit, Pornanong

    2016-01-01

    Sericin was extracted from three strains of Thai Bombyx mori silk cocoons (white shell Chul1/1, greenish shell Chul3/2, and yellow shell Chul4/2) by a high-pressure and high-temperature technique. The characteristics of sericin extracted from different fractions (15, 45, and 60 min extraction process) were compared. No differences in amino acid composition were observed among the three fractions. For all silk strains, sericin extracted from a 15-min process presented the highest molecular weight. The biological potential of the different sericin samples as a bioadditive for 3T3 fibroblast cells was assessed. When comparing sericin extracted from three silk strains, sericin fractions extracted from Chul4/2 improved cell proliferation, while sericin from Chul 1/1 activated Type I collagen production to the highest extent. This study allows the natural variability of sericin obtained from different sources and extraction conditions to be addressed and provides clues for the selection of sericin sources.

  13. Nitric oxide synthase during early embryonic development in silkworm Bombyx mori: Gene expression, enzyme activity, and tissue distribution.

    PubMed

    Kitta, Ryo; Kuwamoto, Marina; Yamahama, Yumi; Mase, Keisuke; Sawada, Hiroshi

    2016-12-01

    To elucidate the mechanism for embryonic diapause or the breakdown of diapause in Bombyx mori, we biochemically analyzed nitric oxide synthase (NOS) during the embryogenesis of B. mori. The gene expression and enzyme activity of B. mori NOS (BmNOS) were examined in diapause, non-diapause, and HCl-treated diapause eggs. In the case of HCl-treated diapause eggs, the gene expression and enzyme activity of BmNOS were induced by HCl treatment. However, in the case of diapause and non-diapause eggs during embryogenesis, changes in the BmNOS activity and gene expressions did not coincide except 48-60 h after oviposition in diapause eggs. The results imply that changes in BmNOS activity during the embryogenesis of diapause and non-diapause eggs are regulated not only at the level of transcription but also post-transcription. The distribution and localization of BmNOS were also investigated with an immunohistochemical technique using antibodies against the universal NOS; the localization of BmNOS was observed mainly in the cytoplasm of yolk cells in diapause eggs and HCl-treated diapause eggs. These data suggest that BmNOS has an important role in the early embryonic development of the B. mori.

  14. Genome-wide identification and analysis of JHBP-domain family members in the silkworm Bombyx mori.

    PubMed

    Li, Wei; Cheng, Tingcai; Hu, Wenbo; Peng, Zhangchuan; Liu, Chun; Xia, Qingyou

    2016-12-01

    Juvenile hormone (JH) regulates the insect growth and development. JH appears in the hemolymph bound by a specific glycoprotein, juvenile hormone-binding protein (JHBP), which serves as a carrier to release the hormone to target tissues and cells. However, JHBP family candidates, expression patterns, and functional implications are still unclear. In this study, we identified 41 genes-containing conserved JHBP domains distributed across eight chromosomes of the silkworm Bombyx mori. A phylogenetic tree showed that the silkworm JHBP (BmJHBP) genes could be classified into two major branches and four subfamilies. Microarray data revealed that BmJHBP genes exhibit various expression patterns and are expressed in different tissues, periods, and sexes. The expression of BmJHBP genes was generally higher in the head, integument, midgut, fat body, testis, and ovary than in the anterior of the silk gland (ASG), median of the silk gland (MSG), posterior of the silk gland (PSG), hemocyte, and Malpighian tubule. BmJHBPd2, in particular, was investigated by Western Blotting, and immunofluorescent assay and was found to be highly expressed in the PSG cytoplasm on day 3 of the fifth instar, coinciding with silk production. Taken together, our findings will be useful in improving understanding the complexity of the JHBP family, and will lay the foundation of explaining functional characterization for further research.

  15. Bombyx mori nucleopolyhedrovirus ORF94, a novel late protein is identified to be a component of ODV structural protein.

    PubMed

    Liang, Guiting; Li, Guohui; Chen, Keping; Yao, Qin; Chen, Huiqing; Zhou, Yang

    2010-09-01

    Orf94 (Bm94) of Bombyx mori nucleopolyhedrovirus (BmNPV) potentially encodes 424-amino acids with a predicted molecular weight of 49.4 kDa, but its function remains unknown. Blast search results revealed that Bm94 homologues exist in 10 completely sequenced Lepidopteron NPVs with identities ranging from 95 to 37%. Results of our recent study showed that Bm94 was transcribed from 12 to 72 h and the corresponding protein was detected from 24 to 72 h post-infection. Furthermore, Western blot analysis revealed that Bm94 was present in occlusion-derived virus (ODV) and in total protein from BmNPV-infected BmN cells, but not in budded virus. Immunofluorescence analysis revealed that the protein located primarily in the cytoplasm and was also present in the nucleus in the later infection. In conclusion, these results together indicated that Bm94 was a late gene, which distributed both in the cytoplasm and in the nucleus, and was identified to be a component of BmNPV ODV.

  16. Bombyx mori nucleopolyhedrovirus ORF79 encodes a 28-kDa structural protein of the ODV envelope.

    PubMed

    Xu, H-J; Yang, Z-N; Wang, F; Zhang, C-X

    2006-04-01

    Open reading frame 79 of Bombyx mori nucleopolyhedrovirus (Bm79) is a conserved gene whose homologues have been identified in all 26 of the completely sequenced baculovirus genomes, including lepidopteran NPVs and GVs, hymenopteran NPVs, and a dipteran baculovirus. Northern blot analysis showed that the Bm79 transcript was about 850 nucleotides long and was initiated 12 h p.i. Temporal expression analysis revealed a 28-kDa protein, which was detected beginning 24 h p.i. using a polyclonal antibody against GST-Bm79 fusion protein. The 28-kDa protein was detected in the occlusion-derived virus envelope (ODV-E), but not in budded viruses. This observation was confirmed by observing ultrathin sections of polyhedra using immunoelectron microscopy. This demonstrated that the protein was present within the nuclei of cells. These results suggest that Bm79 is a functional gene that encodes a structural protein associated with the envelope of occlusion-derived virus (ODV).

  17. The Bombyx mori nucleopolyhedrovirus (BmNPV) ODV-E56 envelope protein is also a per os infectivity factor.

    PubMed

    Xiang, Xingwei; Chen, Lin; Guo, Aiqin; Yu, Shaofang; Yang, Rui; Wu, Xiaofeng

    2011-01-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) odv-e56 gene is a late gene and encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. To determine its role in the BmNPV life cycle, an odv-e56 null virus, BmE56D, was constructed through homologous recombination. A repaired virus was also constructed, named BmE56DR. The production of budded virion (BV) and polyhedra, the replication of viral DNA, and the morphological of infected BmN cells were analyzed, revealing no significant difference among the BmE56D, the wild-type (WT), and the BmE56DR virus. Larval bioassays demonstrated that injection of BmE56D BV into the hemocoel could kill B. mori larvae as efficiently as repaired and WT viruses, however BmE56D was unable to infect the B. mori larvae when inoculated per os. Thus, these results indicated that ODV-E56 envelope protein of BmNPV is also a per os infectivity factor (PIF), but is not essential for virus replication.

  18. Bm91 is an envelope component of ODV but is dispensable for the propagation of Bombyx mori nucleopolyhedrovirus.

    PubMed

    Tang, Qi; Li, Guohui; Yao, Qin; Chen, Liang; Lv, Peng; Lian, Chaoqun; Chen, Keping

    2013-05-01

    Orf91 (Bm91) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene that encodes a predicted 105-amino-acid protein, but its function remains unknown. In the current study, 5'-RACE revealed that the transcription initiation site of Bm91 was - 12 nucleotides upstream of the start codon ATG, transcription of Bm91 was detected from 12 to 96 h postinfection (p.i.) and Bm91 protein was detected from 24 to 96 h p.i. in BmNPV-infected BmN cells. Furthermore, Western blot analysis revealed that Bm91 was in occlusion-derived virus (ODV) but not in budded virus (BV). To investigate the role of Bm91 in baculovirus life cycle, a Bm91-knockout virus was constructed by bacmid recombination in E. coli. Fluorescence and light microscopy showed that the production of BV and occlusion bodies (OBs) in Bm91-deficient-virus-infected BmN cells were similar to those in wild-type-virus-infected ones. Bioassay results showed that genetic deletion of Bm91 did not significantly affect BmNPV infectivity, but extended the median lethal time (LT50). Taken together, these results indicate that Bm91 is not essential for viral propagation in vitro, but absence of the gene may affect the virulence of ODVs in silkworm larvae.

  19. Abortive replication of Bombyx mori nucleopolyhedrovirus in Sf9 and High Five cells: defective nuclear transport of the virions.

    PubMed

    Katou, Yasuhiro; Ikeda, Motoko; Kobayashi, Michihiro

    2006-04-10

    Despite close genetic relationship, Bombyx mori nucleopolyhedrovirus (BmNPV) and Autographa californica multicapsid NPV (AcMNPV) display a distinct host range property. Here, BmNPV replication was examined in Sf9 and High Five cells that were nonproductive for BmNPV infection but supported high titers of AcMNPV replication. Recombinant BmNPV, vBm/gfp/lac, containing bm-ie1 promoter-driven egfp showed that few Sf9 and High Five cells infected with vBm/gfp/lac expressed EGFP, while large proportion of EGFP-expressing cells was observed when transfected with vBm/gfp/lac DNA. Immunocytochemical analysis showed that BmNPV was not imported into the nucleus of these two cell lines, while recombinant BmNPV, vBmDelta64/ac-gp64 possessing AcMNPV gp64 was imported into the nucleus, yielding progeny virions in High Five cells, but not Sf9 cells. These results indicate that the defective nuclear import of infected virions due to insufficient BmNPV GP64 function is involved in the restricted BmNPV replication in Sf9 and High Five cells.

  20. Isolation, Purification, and Identification of an Important Pigment, Sepiapterin, from Integument of the lemon Mutant of the Silkworm, Bombyx mori

    PubMed Central

    Wang, Jing; Wang, Wenjing; Liu, Chaoliang; Meng, Yan

    2013-01-01

    Sepiapterin is the precursor of tetrahydrobiopterin, an important coenzyme of aromatic amino acid hydroxylases, the lack of which leads to a variety of physiological metabolic diseases or neurological syndromes in humans. Sepiapterin is a main pigment component in the integument of the lemon mutant of the silkworm, Bombyx mori (L.) (Lepidoptera: Bombycidae), and is present there in extremely high content, so lemon is a valuable genetic resource to extract sepiapterin. In this study, an effective experimental system was set up for isolation and purification of sepiapterin from lemon silkworms by optimizing homogenization solvent, elution buffer, and separation chromatographic column. The results showed that ethanol was the most suitable solvent to homogenize the integument, with a concentration of 50% and solid:liquid ratio of 1:20 (g/mL). Sepiapterin was purified successively by column chromatography of cellulose Ecteola, sephadex G-25-150, and cellulose phosphate, and was identified by ultraviolet-visible absorption spectrometry. A stable and accurate high performance liquid chromatography method was constructed to identify sepiapterin and conduct qualitative and quantitative analyses. Sepiapterin of high purity was achieved, and the harvest reached about 40 ug/g of integument in the experiments. This work helps to obtaining natural sepiapterin in large amounts in order to use the lemon B. mori mutant to produce BH4 in vitro. PMID:24773269

  1. Antioxidative capacity in the fat body of Bombyx mori is increased following oral administration of 4-methylumbelliferone.

    PubMed

    Fang, Yan; Wang, Hua; Zhu, Wenjuan; Wang, Lu; Liu, Hengjiang; He, Yue; Xu, Xu; Yin, Weimin; Sima, Yanghu; Xu, Shiqing

    2014-01-01

    Plant sources of umbelliferones have tumor-inhibitory effects at the cellular level. However, their physiological functions in animals are largely unresolved. In this study, we provide evidence to show that 4-methylumbelliferone (4-MU) participates in the regulation of antioxidative capacity in the fat body of Bombyx mori, a tissue similar to mammalian liver in this model invertebrate. Larvae (3rd day of the 5th instar) were orally exposed to 4 mM 4-MU, an umbelliferone, which swiftly induced the generation of a large number of ROS (e.g. H2O2 increased 6 to 8-fold), and 4-MU was detected in the fat body 8 min after administration. In addition, the activities of CAT and GPx were up-regulated 4 to 11-fold and 2 to 16-fold, respectively, and were helpful in defending fat body cells against oxidative injury in combination with NADPH. Furthermore, significant increases in the contents of T-AOC (up to approx. 2-fold), antioxidants of ASAFR (by 2 to 4-fold) and GSH were detected.

  2. Physiological requirements for 20-hydroxyecdysone-induced rectal sac distention in the pupa of the silkworm, Bombyx mori.

    PubMed

    Suzuki, Takumi; Sakurai, Sho; Iwami, Masafumi

    2010-06-01

    Successful insect development is achieved via appropriate fluctuation of ecdysteroid levels. When an insect's ecdysteroid level is disrupted, physiological and developmental defects occur. In the pupa of the silkworm, Bombyx mori, the rectal sac is an essential organ that operates as a repository for degraded ecdysteroids, and it can be distended by administration of 20-hydroxyecdysone (20E). Our previous study showed that rectal sac distention appears 4 days after 20E administration. Hemolymph ecdysteroid levels, however, decrease to lower level during this period. Thus, the timing of the rectal sac distention does not match with that of ecdysteroid elevation. Here, we examine how 20E induces rectal sac distention. A ligature experiment and ecdysteroid quantification showed that continuous 20E stimulation induces rectal sac distention. Thorax tissue contributed to the continuous 20E stimulation needed to induce distention. Ecdysteroid released from the thorax tissue may be converted to 20E by ecdysone 20-hydroxylase to produce continuous 20E stimulation. Thus, the ecdysone metabolic pathway plays a critical role in rectal sac distention.

  3. Sex-linked transcription factor involved in a shift of sex-pheromone preference in the silkmoth Bombyx mori

    PubMed Central

    Fujii, Tsuguru; Fujii, Takeshi; Namiki, Shigehiro; Abe, Hiroaki; Sakurai, Takeshi; Ohnuma, Akio; Kanzaki, Ryohei; Katsuma, Susumu; Ishikawa, Yukio; Shimada, Toru

    2011-01-01

    In the sex-pheromone communication systems of moths, odorant receptor (Or) specificity as well as higher olfactory information processing in males should be finely tuned to the pheromone of conspecific females. Accordingly, male sex-pheromone preference should have diversified along with the diversification of female sex pheromones; however, the genetic mechanisms that facilitated the diversification of male preference are not well understood. Here, we explored the mechanisms involved in a drastic shift in sex-pheromone preference in the silkmoth Bombyx mori using spli mutants in which the genomic structure of the gene Bmacj6, which encodes a class IV POU domain transcription factor, is disrupted or its expression is repressed. B. mori females secrete an ∼11:1 mixture of bombykol and bombykal. Bombykol alone elicits full male courtship behavior, whereas bombykal alone shows no apparent activity. In the spli mutants, the behavioral responsiveness of males to bombykol was markedly reduced, whereas bombykal alone evoked full courtship behavior. The reduced response of spli males to bombykol was explained by the paucity of bombykol receptors on the male antennae. It was also found that, in the spli males, neurons projecting into the toroid, a compartment in the brain where bombykol receptor neurons normally project, responded strongly to bombykal. The present study highlights a POU domain transcription factor, Bmacj6, which may have caused a shift of sex-pheromone preference in B. mori through Or gene choice and/or axon targeting. PMID:22006327

  4. Genome-Wide Analysis of Differentially Expressed microRNA in Bombyx mori Infected with Nucleopolyhedrosis Virus.

    PubMed

    Wu, Ping; Jiang, Xiaoxu; Guo, Xijie; Li, Long; Chen, Tao

    2016-01-01

    Bombyx mori nucleopolyhedrosis virus (BmNPV) is a major pathogen that threatens the growth and sustainability of the sericulture industry. Since microRNAs (miRNAs) have been shown to play important roles in host-pathogen interactions, in this study we investigated the effects of BmNPV infection on silkworm microRNAs expression profile. To achieve this, we constructed and deep-sequenced two small RNA libraries generated from BmNPV infected and un-infected larvae. The results revealed that 38 silkworm miRNAs were differentially expressed after BmNPV infection. Based on the GO analysis, their predicted target genes were found to be involved in diverse functions such as binding, catalytic, virion and immune response to stimulus suggesting their potential roles in host-virus interactions. Using the dual-luciferase reporter assay, we confirmed that Bmo-miR-277-5p, up-regulated in BmNPV-infected larvae, targeted the B. mori DNA cytosine-5 methyltransferase (Dnmt2) gene which may play potential role in silkworm-BmNPV interaction. These results provide new insights into exploring the interaction mechanism between silkworm and BmNPV.

  5. Comparison of Similarity Coefficients used for Cluster Analysis with Amplified Fragment Length Polymorphism Markers in the Silkworm, Bombyx mori

    PubMed Central

    Dalirsefat, Seyed Benyamin; da Silva Meyer, Andréia; Mirhoseini, Seyed Ziyaeddin

    2009-01-01

    Establishing accurate genetic similarity and dissimilarity between individuals is an essential and decisive point for clustering and analyzing inter and intra population diversity because different similarity and dissimilarity indices may yield contradictory outcomes. We assessed the variations caused by three commonly used similarity coefficients including Jaccard, Sorensen-Dice and Simple matching in the clustering and ordination of seven Iranian native silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), strains analyzed by amplified fragment length polymorphism markers. Comparisons among the similarity coefficients were made using the Spearman correlation analysis, dendrogram evaluation (visual inspection and consensus fork index - CIC), projection efficiency in a two-dimensional space, and groups formed by the Tocher optimization procedure. The results demonstrated that for almost all methodologies, the Jaccard and Sorensen-Dice coefficients revealed extremely close results, because both of them exclude negative co-occurrences. Due to the fact that there is no guarantee that the DNA regions with negative cooccurrences between two strains are indeed identical, the use of coefficients such as Jaccard and Sorensen-Dice that do not include negative co-occurrences was imperative for closely related organisms. PMID:20050782

  6. Inbreeding Effects on Quantitative Traits in Random Mating and Selected Populations of the Mulberry Silkworm, Bombyx mori

    PubMed Central

    Doreswamy, Jamuna; Gopal, Subramanya

    2012-01-01

    The objective of the present study was to estimate the level of inbreeding coefficient during inbreeding of the pedigree of random mating and selected populations of two distinct races of mulberry silkworm, Bombyx mori (Lepidoptera: Bombycidae), in the silkworm germplasm. The six generation data of the two races, namely multivoltine Pure Mysore and bivoltine NB4D2, were studied for inbreeding depression coefficient using the residual maximum likelihood method, utilizing two statistical models by analyzing six quantitative traits, namely, larval weight, cocoon weight, shell weight, shell ratio, pupation rate, and filament length. The results of the present experiment demonstrated that the inbreeding coefficient was significant in Model 1 for most of the economic traits in the random mating populations of both the races compared to those of selected populations. These results suggest that during stock maintenance, application of rigid selection for increased numbers of generations helps to retain original characteristics of the pure races while reducing the deleterious effects of inbreeding. The significance of inbreeding coefficient is discussed with reference to the inbreeding of silk moths in the silkworm germplasm. PMID:23461728

  7. Genome-Wide Analysis of Differentially Expressed microRNA in Bombyx mori Infected with Nucleopolyhedrosis Virus

    PubMed Central

    Wu, Ping; Jiang, Xiaoxu; Guo, Xijie; Li, Long; Chen, Tao

    2016-01-01

    Bombyx mori nucleopolyhedrosis virus (BmNPV) is a major pathogen that threatens the growth and sustainability of the sericulture industry. Since microRNAs (miRNAs) have been shown to play important roles in host-pathogen interactions, in this study we investigated the effects of BmNPV infection on silkworm microRNAs expression profile. To achieve this, we constructed and deep-sequenced two small RNA libraries generated from BmNPV infected and un-infected larvae. The results revealed that 38 silkworm miRNAs were differentially expressed after BmNPV infection. Based on the GO analysis, their predicted target genes were found to be involved in diverse functions such as binding, catalytic, virion and immune response to stimulus suggesting their potential roles in host-virus interactions. Using the dual-luciferase reporter assay, we confirmed that Bmo-miR-277-5p, up-regulated in BmNPV-infected larvae, targeted the B. mori DNA cytosine-5 methyltransferase (Dnmt2) gene which may play potential role in silkworm-BmNPV interaction. These results provide new insights into exploring the interaction mechanism between silkworm and BmNPV. PMID:27806111

  8. dsRNA interference on expression of a RNA-dependent RNA polymerase gene of Bombyx mori cytoplasmic polyhedrosis virus.

    PubMed

    Pan, Zhong-Hua; Gao, Kun; Hou, Cheng-Xiang; Wu, Ping; Qin, Guang-Xing; Geng, Tao; Guo, Xi-Jie

    2015-07-01

    Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is one of the major viral pathogens in silkworm. Its infection often results in significant losses to sericulture. Studies have demonstrated that RNAi is one of the important anti-viral mechanisms in organisms. In this study, three dsRNAs targeting the RNA-dependent RNA polymerase (RDRP) gene of BmCPV were designed and synthesized with 2'-F modification to explore their interference effects on BmCPV replication in silkworm larvae. The results showed that injecting dsRNA in the dosage of 4-6 ng per mg body weight into the 5th instar larvae can interfere with the BmCPV-RDRP expression by 93% after virus infection and by 99.9% before virus infection. In addition, the expression of two viral structural protein genes (genome RNA segments 1 and 5) was also decreased with the decrease of RDRP expression, suggesting that RNAi interference of BmCPV-RDRP expression could affect viral replication. The study provides an effective method for investigating virus replication as well as the virus-host interactions in the silkworm larvae using dsRNA. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. piggyBac transposon-derived targeting shRNA interference against the Bombyx mori nucleopolyhedrovirus (BmNPV).

    PubMed

    Zhou, Fang; Chen, Rui-ting; Lu, Yan; Liang, Shuang; Wang, Mei-xian; Miao, Yun-gen

    2014-12-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most destructive diseases in silkworm, which has caused the main damage to sericulture industry. In this study, we developed a system of RNAi to prevent the BmNPV infection using the piggyBac transposon-derived targeting short hairpin RNA (shRNA) interference. The shRNAs targeting the genes of i.e.-1, lef-1, lef-2 and lef-3 of BmNPV were designed and used to inhibit the intracellular replication or multiplication of BmNPV in Bm cells. The highest activity was presented in the shRNA targeting the i.e.-1c of BmNPV, of which the inhibition rate reached 94.5 % in vitro. Further a stable Bm cell line of piggyBac transposon-derived targeting shRNA interference against BmNPV was established, which has a highly efficacious suppression on virus proliferation. These results indicated that the recombinant shRNA expression system was a useful tool for resistance to BmNPV in vitro. The approach by recombinant shRNAs opens a door of RNAi technology as a strategy that offering technically simpler, cheaper, and quicker gene knockdown for promising research and biotechnology application on silkworm lethal diseases.

  10. Phylogenetic analysis of Bombyx mori nucleopolyhedrovirus polyhedrin and p10 genes in wild isolates from Guangxi Zhuang Autonomous Region, China.

    PubMed

    Liang, Xiang; Lu, Zhuan-Ling; Wei, Bing-Xing; Feng, Jian-Ling; Qu, Dacai; Luo, Ting Rong

    2013-02-01

    Bombyx mori nucleopolyhedrovirus (BmNPV) is a severe pathogen that seriously impacts the sericulture industry. In this study, 45 wild BmNPV isolates were collected from different silkworm-raising regions in China's Guangxi Zhuang Autonomous Region. Two highly expressed very late genes from each isolate, polyhedrin and p10, were sequenced and subjected to phylogenetic analysis. The polyhedrin gene was found to be highly conserved, while the p10 gene was more variable frequently harboring point mutations and displaying variations in codon use without obvious codon bias. The BmNPV isolates from Guangxi were separated into three main clades, I, II, and III, according to the p10 gene phylogenetic tree. The geographical distribution of clade I isolates in Guangxi showed a concentrated pattern and that of clade II isolates showed a connected pattern. Clade III isolates were irregularly scattered throughout Guangxi. Local transmission of this pathogen clearly occurred in the silkworm-raising regions in Guangxi. This study may provide some data on BmNPV transmission in the silkworm-raising regions and be helpful in devising strategies for the prevention and control of BmNPV disease.

  11. Bm59 is an early gene, but is unessential for the propagation and assembly of Bombyx mori nucleopolyhedrovirus.

    PubMed

    Hu, Xiaolong; Shen, Yunwang; Zheng, Qin; Wang, Guobao; Wu, Xiaofeng; Gong, Chengliang

    2016-02-01

    Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen that specifically infects the domestic silkworm and causes serious economic loss to sericulture around the world. The function of BmNPV Bm59 gene in the viral life cycle is inconclusive. To investigate the role of Bm59 during viral infection, the transcription initiation site and temporal expression of Bm59 were analyzed, and Bm59-knockout virus was generated through homologous recombination in Escherichia coli. The results showed that Bm59 is an early transcription gene with an atypia early transcriptional start motif. Budded virion (BV) production and DNA replication in the BmN cells transfected with the Bm59-knockout virus bacmid were similar to those in the cells transfected with the wild-type virus. Electron microscopy revealed that the occlusion-derived virus can be produced in cells infected with the Bm59-knockout virus. These results indicated that Bm59 is an early gene and is not essential for viral replication or assembly of BmNPV. These findings suggested that non-essential gene (Bm59) remained in the viral genome, which may interact with other viral/host genes in a certain situation.

  12. Amplified Fragment Length Polymorphism Mapping of Quantitative Trait Loci for Economically Important Traits in the Silkworm, Bombyx mori

    PubMed Central

    Mirhoseini, Seyed Z; Rabiei, Babak; Potki, Payam; Dalirsefat, Seyed B

    2010-01-01

    Cocoon related characteristics are economically important traits in the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). In this study a genetic linkage map was developed that identified QTL controlling the cocoon weight, cocoon shell weight, and cocoon shell percentage using 161 amplified fragment length polymorphism (AFLP) markers. Twenty PstI/TaqI primer combinations were employed to genotype 78 F2 progenies derived from a cross between P107 Japanese inbred line and Khorasan Lemon Iranian native strain. Among polymorphic markers, 159 AFLP markers were assigned to 24 linkage groups at the LOD threshold of 2.5 that varied in length from 4 to 299 cM. The total length of the linkage map was 2747 cM, giving an average marker resolution of 19.31 cM. A total of 21 AFLP markers were identified that were distributed over the ten linkage groups linked to the three studied traits using the composite interval mapping method. The explained variation rate by QTL controlling cocoon weight, cocoon shell weight, and cocoon shell percentage ranged from 0.02% to 64.85%, 0.2% to 49.11%, and 0.04% to 84.20%, respectively. These QTL controlled by different actions as well as under dominance, additive, partial dominance, dominance, and over dominance. PMID:21070171

  13. 20-Hydroxyecdysone (20E) Primary Response Gene E75 Isoforms Mediate Steroidogenesis Autoregulation and Regulate Developmental Timing in Bombyx*

    PubMed Central

    Li, Kang; Tian, Ling; Guo, Zhongjian; Guo, Sanyou; Zhang, Jianzhen; Gu, Shi-Hong; Palli, Subba R.; Cao, Yang; Li, Sheng

    2016-01-01

    The temporal control mechanisms that precisely control animal development remain largely elusive. The timing of major developmental transitions in insects, including molting and metamorphosis, is coordinated by the steroid hormone 20-hydroxyecdysone (20E). 20E involves feedback loops to maintain pulses of ecdysteroid biosynthesis leading to its upsurge, whereas the underpinning molecular mechanisms are not well understood. Using the silkworm Bombyx mori as a model, we demonstrated that E75, the 20E primary response gene, mediates a regulatory loop between ecdysteroid biosynthesis and 20E signaling. E75 isoforms A and C directly bind to retinoic acid receptor-related response elements in Halloween gene promoter regions to induce gene expression thus promoting ecdysteroid biosynthesis and developmental transition, whereas isoform B antagonizes the transcriptional activity of isoform A/C through physical interaction. As the expression of E75 isoforms is differentially induced by 20E, the E75-mediated regulatory loop represents a fine autoregulation of steroidogenesis, which contributes to the precise control of developmental timing. PMID:27365399

  14. 20-Hydroxyecdysone (20E) Primary Response Gene E75 Isoforms Mediate Steroidogenesis Autoregulation and Regulate Developmental Timing in Bombyx.

    PubMed

    Li, Kang; Tian, Ling; Guo, Zhongjian; Guo, Sanyou; Zhang, Jianzhen; Gu, Shi-Hong; Palli, Subba R; Cao, Yang; Li, Sheng

    2016-08-26

    The temporal control mechanisms that precisely control animal development remain largely elusive. The timing of major developmental transitions in insects, including molting and metamorphosis, is coordinated by the steroid hormone 20-hydroxyecdysone (20E). 20E involves feedback loops to maintain pulses of ecdysteroid biosynthesis leading to its upsurge, whereas the underpinning molecular mechanisms are not well understood. Using the silkworm Bombyx mori as a model, we demonstrated that E75, the 20E primary response gene, mediates a regulatory loop between ecdysteroid biosynthesis and 20E signaling. E75 isoforms A and C directly bind to retinoic acid receptor-related response elements in Halloween gene promoter regions to induce gene expression thus promoting ecdysteroid biosynthesis and developmental transition, whereas isoform B antagonizes the transcriptional activity of isoform A/C through physical interaction. As the expression of E75 isoforms is differentially induced by 20E, the E75-mediated regulatory loop represents a fine autoregulation of steroidogenesis, which contributes to the precise control of developmental timing. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. The non-structural protein NS-2 of Bombyx mori parvo-like virus is localized to the nuclear membrane.

    PubMed

    Wang, Fenghua; Hu, Zhaoyang; He, Yuanqing; Li, Guohui; Kong, Jie; Cao, Jian; Chen, Keping; Yao, Qin

    2011-07-01

    Bombyx mori parvo-like virus (BmPLV) has two complementary single-stranded DNA genome (VD1 and VD2) and owns a self-encoding DNA polymerase motif, but its replication mechanism is unclear. In our previous research, a protein encoded by VD1-ORF1 was indentified in the midgut of BmPLV China Zhenjiang isolate-(BmPLV-Z) infected silkworm larvae via two-dimensional gel electrophoresis (2-DE). This protein was named as non-structural protein 2 (NS2), which showed no similarity to that of parvoviruses. To date, little is known about it. In this study, sequence alignment results showed that NS2 shared homology with some chromosomal replication initiator protein dnaA and DNA-binding response regulators. The ns2 was cloned and expressed in E. coli, and then a polyclonal antibody of the NS2 protein was prepared successfully. The data from real-time quantitative PCR displayed that the transcription of VD1-ORF1 from BmPLV-Z-infected midguts started from 28-h post inoculation (h p.i.) in low amounts, but in high amounts at late stages of infection. Immunofluorescence showed that NS2 ultimately concentrated on the nuclear membrane in BmN cells at late stages, indicating that NS2 might be associated with integral membrane protein.

  16. Involvement of a bifunctional fatty-acyl desaturase in the biosynthesis of the silkmoth, Bombyx mori, sex pheromone

    PubMed Central

    Moto, Ken'ichi; Suzuki, Masataka G.; Hull, J. Joe; Kurata, Ryuichiro; Takahashi, Shunya; Yamamoto, Masanobu; Okano, Kazuhiro; Imai, Kiyohiro; Ando, Tetsu; Matsumoto, Shogo

    2004-01-01

    The straight-chain C10 to C18 unsaturated aliphatic compounds containing an oxygenated functional group (aldehyde, alcohol, or acetate ester) derived from saturated C16 or C18 fatty acids are a major class of sex pheromone components produced by female moths. In the biosynthesis of these pheromone components, various combinations of limited chain-shortening and regio- and stereospecific desaturation reactions significantly contribute to the production of a vast number of the species-specific pheromone components in Lepidoptera. Biosynthesis of the silkmoth sex pheromone bombykol, (E,Z)-10,12-hexadecadien-1-ol, involves two consecutive desaturation steps, the second of which is unique in that it generates a conjugated diene system from the Δ11-monoene C16 intermediate. In experiments designed to characterize the acyl-CoA desaturases responsible for bombykol biosynthesis, we have cloned three cDNAs encoding desaturase family members from the pheromone gland of the inbred strain of the silkmoth, Bombyx mori. Transcript analyses by RT-PCR and subsequent functional assays using a Bac-to-Bac baculovirus expression system revealed that desat1 is the only desaturase gene prominently expressed during pheromonogenesis and that its gene product, B. mori Desat1, possesses both Z11 desaturation and Δ10,12-desaturation activities. Consequently, we have concluded that B. mori Desat1 is not only a bifunctional desaturase involved in bombykol biosynthesis but that it is also the enzyme responsible for both desaturation steps. PMID:15173596

  17. The midgut of the silkmoth Bombyx mori is able to recycle molecules derived from degeneration of the larval midgut epithelium.

    PubMed

    Franzetti, Eleonora; Romanelli, Davide; Caccia, Silvia; Cappellozza, Silvia; Congiu, Terenzio; Rajagopalan, Muthukumaran; Grimaldi, Annalisa; de Eguileor, Magda; Casartelli, Morena; Tettamanti, Gianluca

    2015-08-01

    The midgut represents the middle part of the alimentary canal and is responsible for nutrient digestion and absorption in insect larva. Despite the growing interest in this organ for different purposes, such as studies on morphogenesis and differentiation, stem cell biology, cell death processes and transport mechanisms, basic information on midgut development is still lacking for a large proportion of insect species. Undoubtedly, this lack of data could hinder the full exploitation of practical applications that involve midgut as their primary target. This may represent in particular a significant problem for Lepidoptera, an insect order that includes some of the most important species of high economic importance. With the aim of overcoming this fragmentation of knowledge, we performed a detailed morphofunctional analysis of the midgut of the silkworm, Bombyx mori, a representative model among Lepidoptera, during its development from the larval up to the adult stage, focusing attention on stem cells. Our data demonstrate stem cell proliferation and differentiation, not only in the larval midgut but also in the pupal and adult midgut epithelium. Moreover, we present evidence for a complex trophic relationship between the dying larval epithelium and the new adult one, which is established during metamorphosis. This study, besides representing the first morphological and functional characterization of the changes that occur in the midgut of a lepidopteron during the transition from the larva to the moth, provides a detailed analysis of the midgut of the adult insect, a stage that has been neglected up to now.

  18. Developmental and sex-specific differences in expression of neuropeptides derived from allatotropin gene in the silkmoth Bombyx mori.

    PubMed

    Bednár, Branislav; Roller, Ladislav; Čižmár, Daniel; Mitrová, Diana; Žitňan, Dušan

    2017-05-01

    Allatotropin (AT) and related neuropeptides are widespread bioactive molecules that regulate development, food intake and muscle contractions in insects and other invertebrates. In moths, alternative splicing of the at gene generates three mRNA precursors encoding AT with different combinations of three structurally similar AT-like peptides (ATLI-III). We used in situ hybridization and immunohistochemistry to map the differential expression of these transcripts during the postembryonic development of Bombyx mori. Transcript encoding AT alone was expressed in numerous neurons of the central nervous system and frontal ganglion, whereas transcripts encoding AT with ATLs were produced by smaller specific subgroups of neurons in larval stages. Metamorphosis was associated with considerable developmental changes and sex-specific differences in the expression of all transcripts. The most notable was the appearance of AT/ATL transcripts (1) in the brain lateral neurosecretory cells producing prothoracicotropic hormone; (2) in the male-specific cluster of about 20 neurons in the posterior region of the terminal abdominal ganglion; (3) in the female-specific medial neurons in the abdominal ganglia AG2-7. Immunohistochemical staining showed that these neurons produced a mixture of various neuropeptides and innervated diverse peripheral organs. Our data suggest that AT/ATL neuropeptides are involved in multiple stage- and sex-specific functions during the development of B. mori.

  19. [Effects of sub-lethal dosages abamectin on food intake and digestive enzyme activities of silkworm Bombyx mori L].

    PubMed

    Zhu, Jiu-sheng; Wang, Jing; Gao, Hai-yan; Qin, Shu; Qiao, Xiong-wu; Han, Ju-cai

    2008-11-01

    Mulberry leaves treated with sub-lethal dosages (LC5, LC10 and LC20) abameetin were fed to the 5th instar larvae of silkworm (Bombyx mori L.), and the food intake and digestive enzyme activities of the larvae were studied by using gravimetric method and measuring enzyme activities. The results showed that sub-lethal dosages abameetin significantly inhibited the growth and food intake of the larvae, with their body mass and its increase rate as well as their relative growth rate being significantly lower than the control, and accompanied with the decreases of food intake, its relative consumption rate, and feces amount. The efficiency of the conversion of ingested food (ECI) and that of the conversion of digested food (EDI) also reduced, but the approximate digestibility (AD) increased significantly. The amylase and sucrase activities in the midgut of the larvae treated with abameetin decreased significantly for a longer time at the beginning, and then recovered to the same as or a higher level than the control, whereas the trehalase activity decreased significantly for a shorter time at the beginning, then increased significantly, and finally recovered to the normal. It was suggested that sub-lethal dosages abameetin had definite toxicity to the silkworm, and the toxic effect was increased with increasing dosage, which could result in the turbulence of silkworm's digestive system, and further, affect its food intake and its growth and development.

  20. A megalin-like receptor is involved in protein endocytosis in the midgut of an insect (Bombyx mori, Lepidoptera).

    PubMed

    Casartelli, M; Cermenati, G; Rodighiero, S; Pennacchio, F; Giordana, B

    2008-10-01

    The mechanism responsible for fluorescein isothiocyanate (FITC)-albumin internalization by columnar cells in culture obtained from the midgut of Bombyx mori larvae was examined by confocal laser scanning microscopy. Protein uptake changed over time, and it appeared to be energy dependent, since it was strongly reduced by both low temperatures and metabolic inhibitors. Labeled albumin uptake as a function of increasing protein concentration showed a saturation kinetics with a Michaelis constant value of 2.0 +/- 0.6 microM. These data are compatible with the occurrence of receptor-mediated endocytosis. RT-PCR analysis and colocalization experiments with an anti-megalin primary antibody indicated that the receptor involved was a putative homolog of megalin, the multiligand endocytic receptor belonging to the low-density lipoprotein receptor family, responsible for the uptake of various molecules, albumin included, in many epithelial cells of mammals. This insect receptor, like the mammalian counterpart, required Ca(2+) for albumin internalization and was inhibited by gentamicin. FITC-albumin internalization was clathrin mediated, since two inhibitors of this process caused a significant reduction of the uptake, and clathrin and albumin colocalized in the intermicrovillar areas of the apical plasma membrane. The integrity of actin and microtubule organization was essential for the correct functioning of the endocytic machinery.

  1. Diapause specific gene expression in the eggs of multivoltine silkworm Bombyx mori, identified by suppressive subtractive hybridization.

    PubMed

    Sasibhushan, Sirigineedi; Ponnuvel, Kangayam M; Vijayaprakash, Nanjappa B

    2012-04-01

    Molecular mechanism controlling egg diapause remains obscure in silkworm, Bombyx mori. An attempt is made to decipher various molecular events occurring during embryonic diapause in multivoltine silkworm, B. mori. Using suppressive subtractive hybridization (SSH), 186 cDNA clones isolated from both diapause and nondiapause eggs were sequenced. Of the sequenced clones, 29 matched with silkbase entries and these identified putative genes were classified into six functional groups such as regulatory, food utilization, stress response, metabolic, ribosomal and transposable elements. Among these genes, twelve belonged to regulatory group while, one taste receptor type 2 member 117 gene was related to food utilization. One heat shock cognate 70 kDa protein and 3 of the ubiquitin family were identified under stress response category. Similarly, four genes were identified as metabolic genes, 3 belonging to chitin family and one propanediol utilization protein. Of the seven genes identified in ribosomal groups, most of them were 60s ribosomal protein subunits. However, one negative regulation of transcription gene identified was a transposable element. The qPCR analysis confirmed the expression of 21 of the above genes, wherein, 6 were upregulated during diapause, 12 during non-diapause, while, 3 remained unchanged. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Glycerol-3-phosphate O-acyltransferase is required for PBAN-induced sex pheromone biosynthesis in Bombyx mori

    PubMed Central

    Du, Mengfang; Liu, Xiaoguang; Liu, Xiaoming; Yin, Xinming; Han, Shuangyin; Song, Qisheng; An, Shiheng

    2015-01-01

    Female moths employ their own pheromone blends as a communicational medium in mating behavior. The biosynthesis and release of sex pheromone in female moths are regulated by pheromone biosynthesis activating neuropeptide (PBAN) and the corresponding action of PBAN has been well elucidated in Bombyx mori. However, very little is known about the molecular mechanism regarding the biosynthesis of sex pheromone precursor. In this study, quantitative proteomics was utilized to comprehensively elucidate the expression dynamics of pheromone glands (PGs) during development. Proteomic analysis revealed a serial of differentially expressed sex pheromone biosynthesis-associated proteins at the different time points of B. mori development. Most interestingly B. mori glycerol-3-phosphate O-acyltransferase (BmGPAT) was found to be expressed during the key periods of sex pheromone biosynthesis. RNAi knockdown of BmGPAT confirmed the important function of this protein in the biosynthesis of sex pheromone precursor, triacylglcerol (TAG), and subsequently PBAN-induced production of sex pheromone, bombykol. Behavioral analysis showed that RNAi knockdown of GPAT significantly impaired the ability of females to attract males. Our findings indicate that GPAT acts to regulate the biosynthesis of sex pheromone precursor, TAG, thus influencing PBAN-induced sex pheromone production and subsequent mating behavior. PMID:25630665

  3. Insulin-like and IGF-like peptides in the silkmoth Bombyx mori: discovery, structure, secretion, and function

    PubMed Central

    Mizoguchi, Akira; Okamoto, Naoki

    2013-01-01

    A quarter of a century has passed since bombyxin, the first insulin-like peptide identified in insects, was discovered in the silkmoth Bombyx mori. During these years, bombyxin has been studied for its structure, genes, distribution, hemolymph titers, secretion control, as well as physiological functions, thereby stimulating a wide range of studies on insulin-like peptides in other insects. Moreover, recent studies have identified a new class of insulin family peptides, IGF-like peptides, in B. mori and Drosophila melanogaster, broadening the base of the research area of the insulin-related peptides in insects. In this review, we describe the achievements of the studies on insulin-like and IGF-like peptides mainly in B. mori with short histories of their discovery. Our emphasis is that bombyxins, secreted by the brain neurosecretory cells, regulate nutrient-dependent growth and metabolism, whereas the IGF-like peptides, secreted by the fat body and other peripheral tissues, regulate stage-dependent growth of tissues. PMID:23966952

  4. Refinement of ectopic protein expression through the GAL4/UAS system in Bombyx mori: application to behavioral and developmental studies.

    PubMed

    Hara, Chiho; Morishita, Koudai; Takayanagi-Kiya, Seika; Mikami, Akihisa; Uchino, Keiro; Sakurai, Takeshi; Kanzaki, Ryohei; Sezutsu, Hideki; Iwami, Masafumi; Kiya, Taketoshi

    2017-09-18

    Silkmoth, Bombyx mori, is one of the important model insects in which transgenic techniques and the GAL4/UAS system are applicable. However, due to cytotoxicity and low transactivation activity of GAL4, effectiveness of the GAL4/UAS system and its application in B. mori are still limited. In the present study, we refined the previously reported UAS vector by exploiting transcriptional and translational enhancers, and achieved 200-fold enhancement of reporter GFP fluorescence in the GAL4/UAS system. Enhanced protein expression of membrane-targeted GFP and calcium indicator protein (GCaMP5G) drastically improved visualization of fine neurite structures and neural activity, respectively. Also, with the refined system, we generated a transgenic strain that expresses tetanus toxin light chain (TeTxLC), which blocks synaptic transmission, under the control of GAL4. Ectopic TeTxLC expression in the sex pheromone receptor neurons inhibited male courtship behavior, proving effectiveness of TeTxLC on loss-of-function analyses of neural circuits. In addition, suppression of prothoracicotropic hormone (PTTH) or insulin-like peptide (bombyxin) secretion impaired developmental timing and growth rate, respectively. Furthermore, we revealed that larval growth is sex-differentially regulated by these peptide hormones. The present study provides important technical underpinnings of transgenic approaches in silkmoths and insights into mechanisms of postembryonic development in insects.

  5. Orthologs of Human Disease Associated Genes and RNAi Analysis of Silencing Insulin Receptor Gene in Bombyx mori

    PubMed Central

    Zhang, Zan; Teng, Xiaolu; Chen, Maohua; Li, Fei

    2014-01-01

    The silkworm, Bombyx mori L., is an important economic insect that has been domesticated for thousands of years to produce silk. It is our great interest to investigate the possibility of developing the B. mori as human disease model. We searched the orthologs of human disease associated genes in the B. mori by bi-directional best hits of BLAST and confirmed by searching the OrthoDB. In total, 5006 genes corresponding to 1612 kinds of human diseases had orthologs in the B. mori, among which, there are 25 genes associated with diabetes mellitus. Of these, we selected the insulin receptor gene of the B. mori (Bm-INSR) to study its expression in different tissues and at different developmental stages and tissues. Quantitative PCR showed that Bm-INSR was highly expressed in the Malpighian tubules but expressed at low levels in the testis. It was highly expressed in the 3rd and 4th instar larvae, and adult. We knocked down Bm-INSR expression using RNA interference. The abundance of Bm-INSR transcripts were dramatically reduced to ~4% of the control level at 6 days after dsRNA injection and the RNAi-treated B. mori individuals showed apparent growth inhibition and malformation such as abnormal body color in black, which is the typical symptom of diabetic patients. Our results demonstrate that B. mori has potential use as an animal model for diabetic mellitus research. PMID:25302617

  6. The light cycle controls the hatching rhythm in Bombyx mori via negative feedback loop of the circadian oscillator.

    PubMed

    Tao, Hui; Li, Xue; Qiu, Jian-Feng; Liu, Heng-Jiang; Zhang, Da-Yan; Chu, Feng; Sima, Yanghu; Xu, Shi-Qing

    2017-10-01

    Hatching behavior is a key target in silkworm (Bombyx mori) rearing, especially for the control of Lepidoptera pests. According to previous research, hatching rhythms appear to be controlled by a clock mechanism that restricts or "gates" hatching to a particular time. However, the underlying mechanism remains elusive. Under 12-h light:12-h dark photoperiod (LD) conditions, the transcriptional levels of the chitinase5 (Cht5) and hatching enzyme-like (Hel) genes, as well as the enzymatic activities of their gene products, oscillated in time with ambient light cycles, as did the transcriptional levels of the cryptochrome 1, cryptochrome 2, period (per), and timeless genes, which are key components of the negative feedback loop of the circadian rhythm. These changes were related to the expression profile of the ecdysteroid receptor gene and the hatching behavior of B. mori eggs. However, under continuous light or dark conditions, the hatching behavior, the expression levels of Cht5 and Hel, as well as the enzymatic activities of their gene products, were not synchronized unlike under LD conditions. In addition, immunohistochemistry experiments showed that light promoted the translocation of PER from the cytoplasm to the nucleus. In conclusion, LD cycles regulate the hatching rhythm of B. mori via negative feedback loop of the circadian oscillator. © 2017 Wiley Periodicals, Inc.

  7. Pheromone discrimination by a pH-tuned polymorphism of the Bombyx mori pheromone-binding protein.

    PubMed

    Damberger, Fred F; Michel, Erich; Ishida, Yuko; Leal, Walter S; Wüthrich, Kurt

    2013-11-12

    The Bombyx mori pheromone-binding protein (BmorPBP) is known to adopt two different conformations. These are BmorPBP(A), where a regular helix formed by the C-terminal dodecapeptide segment, α7, occupies the ligand-binding cavity, and BmorPBP(B), where the binding site is free to accept ligands. NMR spectra of delipidated BmorPBP solutions at the physiological pH of the bulk sensillum lymph near pH 6.5 show only BmorPBP(A), and in mixtures, the two species are in slow exchange on the chemical shift frequency scale. This equilibrium has been monitored at variable pH and ligand concentrations, demonstrating that it is an intrinsic property of BmorPBP that is strongly affected by pH variation and ligand binding. This polymorphism tunes BmorPBP for optimal selective pheromone transport: Competition between α7 and lipophilic ligands for its binding cavity enables selective uptake of bombykol at the pore endings in the sensillum wall, whereas compounds with lower binding affinity can only be bound in the bulk sensillum lymph. After transport across the bulk sensillum lymph into the lower pH area near the dendritic membrane surface, bombykol is ejected near the receptor, whereas compounds with lower binding affinity are ejected before reaching the olfactory receptor, rendering them susceptible to degradation by enzymes present in the sensillum lymph.

  8. Transcriptome analysis of the Bombyx mori fat body after constant high temperature treatment shows differences between the sexes.

    PubMed

    Wang, Hua; Fang, Yan; Wang, Lipeng; Zhu, Wenjuan; Ji, Haipeng; Wang, Haiying; Xu, Shiqing; Sima, Yanghu

    2014-09-01

    Ambient temperature plays a large role in insect growth, development and even their distribution. The elucidation of the associated molecular mechanism that underlies the effect of constant high temperature will enables us to further understand the stress responses. We constructed four digital gene expression libraries from the fat body of female and male Bombyx mori. Differential gene expression was analyzed after constant high temperature treatment. The results showed that there were significant changes to the gene expression in the fat body after heat treatment, especially in binding, catalytic, cellular and metabolic processes. Constant high temperature may induce more traditional cryoprotectants, such as glycerol, glycogen, sorbitol and lipids, to protect cells from damage, and induce heat oxidative stress in conjunction with the heat shock proteins. The data also indicated a difference between males and females. The heat shock protein-related genes were up-regulated in both sexes but the expression of Hsp25.4 and DnaJ5 were down-regulated in the male fat body of B. mori. This is the first report of such a result. Constant high temperature also affected the expression of other functional genes and differences were observed between male and female fat bodies in the expression of RPS2, RPL37A and MREL. These findings provide abundant data on the effect of high temperature on insects at the molecular level. The data will also be beneficial to the study of differences between the sexes, manifested in variations in gene expression under high temperature.

  9. Mapping and recombination analysis of two moth colour mutations, Black moth and Wild wing spot, in the silkworm Bombyx mori

    PubMed Central

    Ito, K; Katsuma, S; Kuwazaki, S; Jouraku, A; Fujimoto, T; Sahara, K; Yasukochi, Y; Yamamoto, K; Tabunoki, H; Yokoyama, T; Kadono-Okuda, K; Shimada, T

    2016-01-01

    Many lepidopteran insects exhibit body colour variations, where the high phenotypic diversity observed in the wings and bodies of adults provides opportunities for studying adaptive morphological evolution. In the silkworm Bombyx mori, two genes responsible for moth colour mutation, Bm and Ws, have been mapped to 0.0 and 14.7 cM of the B. mori genetic linkage group 17; however, these genes have not been identified at the molecular level. We performed positional cloning of both genes to elucidate the molecular mechanisms that underlie the moth wing- and body-colour patterns in B. mori. We successfully narrowed down Bm and Ws to ~2-Mb-long and 100-kb-long regions on the same scaffold Bm_scaf33. Gene prediction analysis of this region identified 77 candidate genes in the Bm region, whereas there were no candidate genes in the Ws region. Fluorescence in-situ hybridisation analysis in Bm mutant detected chromosome inversion, which explains why there are no recombination in the corresponding region. The comparative genomic analysis demonstrated that the candidate regions of both genes shared synteny with a region associated with wing- and body-colour variations in other lepidopteran species including Biston betularia and Heliconius butterflies. These results suggest that the genes responsible for wing and body colour in B. mori may be associated with similar genes in other Lepidoptera. PMID:26219230

  10. An efficient strategy for producing a stable, replaceable, highly efficient transgene expression system in silkworm, Bombyx mori

    PubMed Central

    Long, Dingpei; Lu, Weijian; Zhang, Yuli; Bi, Lihui; Xiang, Zhonghuai; Zhao, Aichun

    2015-01-01

    We developed an efficient strategy that combines a method for the post-integration elimination of all transposon sequences, a site-specific recombination system, and an optimized fibroin H-chain expression system to produce a stable, replaceable, highly efficient transgene expression system in the silkworm (Bombyx mori) that overcomes the disadvantages of random insertion and post-integration instability of transposons. Here, we generated four different transgenic silkworm strains, and of one the transgenic strains, designated TS1-RgG2, with up to 16% (w/w) of the target protein in the cocoons, was selected. The subsequent elimination of all the transposon sequences from TS1-RgG2 was completed by the heat-shock-induced expression of the transposase in vivo. The resulting transgenic silkworm strain was designated TS3-g2 and contained only the attP-flanked optimized fibroin H-chain expression cassette in its genome. A phiC31/att-system-based recombinase-mediated cassette exchange (RMCE) method could be used to integrate other genes of interest into the same genome locus between the attP sites in TS3-g2. Controlling for position effects with phiC31-mediated RMCE will also allow the optimization of exogenous protein expression and fine gene function analyses in the silkworm. The strategy developed here is also applicable to other lepidopteran insects, to improve the ecological safety of transgenic strains in biocontrol programs. PMID:25739894

  11. Evaluation of menstrual blood stem cells seeded in biocompatible Bombyx mori silk fibroin scaffold for cardiac tissue engineering.

    PubMed

    Rahimi, Maryam; Mohseni-Kouchesfehani, Homa; Zarnani, Amir-Hassan; Mobini, Sahba; Nikoo, Shohreh; Kazemnejad, Somaieh

    2014-08-01

    Recently, silk fibroin scaffolds have been introduced as novel and promising biomaterials in the field of cardiac tissue engineering. This study was designed to compare infiltration, proliferation, and cardiac differentiation potential of menstrual blood-derived stem cells (MenSCs) versus bone marrow-derived mesenchymal stem cells (BMSCs) in Bombyx mori-derived silk scaffold. Our primary data revealed that the fabricated scaffold has mechanical and physical qualities suitable for cardiac tissue engineering. The MenSCs tracking in scaffolds using immunofluorescent staining and scanning electron microscopy confirmed MenSCs attachment, penetration, and distribution within the porous scaffold matrix. Based on proliferation assay using propidium iodide DNA quantification, the significantly higher level of growth rates of both MenSCs and BMSCs was documented in scaffolds than that in two-dimensional culture (p < 0.01). The expression level of TNNT2, a bona fide cardiac differentiation marker, in BMSCs differentiated on silk scaffolds was markedly higher than those cultured in two-dimensional culture indicating the improvement of cardiac differentiation in the silk scaffolds. Furthermore, differentiated MenSCs exhibited higher expression of TNNT2 compared with induced BMSCs. It seems that silk scaffold-seeded MenSCs could be viewed as a novel, safe, natural, and accessible construct for cardiac tissue engineering. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  12. IMPACT OF CYCLICAL CHANGES IN TEMPERATURE ON CIRCADIAN CLOCK GENES EXPRESSION IN Bombyx BmN CELLS.

    PubMed

    Chu, Feng; Qiu, Jian-Feng; Tao, Hui; Li, Xue; Shu, Mei-Ying; Liu, Heng-Jiang; SiMa, Yanghu; Xu, Shiqing

    2016-03-01

    The physiology and metabolism of poikilothermic insects are under the control of environmental temperature. Temperature is the primary cue for the circadian rhythm. Reports on the timing mechanisms of temperature in lepidopterans are limited. This study used Bombyx mori BmN ovarian cells to investigate the effect of temperature on expression of the main circadian clock genes in a negative feedback loop. A 37°C, 30-min high-temperature stimulation induced transcription of the circadian clock genes Cry1, Cry2, Per, and Tim. The gene expression profiles showed rhythmic oscillations, with shortened oscillatory periods for Cry1 and altered oscillatory phases for Cry1 and Per. Cyclical increases in temperature of 2°C starting at 26°C, 5°C starting at 20 or 25°C, or 10°C starting at 20°C induced cyclical changes in expression and protein from the four circadian clock genes. Cyclical temperature changes with a difference of 10°C had the most influence. In conclusion, cyclical changes in temperature with differences from 2 to 10°C reset and synchronized the circadian clock of silkworm BmN cells. Transcription of the genes for and protein from Cry2 and Per showed a better reset and synchronization with cyclical temperature changes. © 2016 Wiley Periodicals, Inc.

  13. Comparative proteomics reveal diverse functions and dynamic changes of Bombyx mori silk proteins spun from different development stages.

    PubMed

    Dong, Zhaoming; Zhao, Ping; Wang, Chen; Zhang, Yan; Chen, Jianping; Wang, Xin; Lin, Ying; Xia, Qingyou

    2013-11-01

    Silkworms (Bombyx mori) produce massive amounts of silk proteins to make cocoons during the final stages of larval development. Although the major components, fibroin and sericin, have been the focus for a long time, few researchers have realized the complexity of the silk proteome. We collected seven kinds of silk fibers spun by silkworm larvae at different developmental stages: the silks spun by new hatched larvae, second instar day 0 larvae, third instar day 0 larvae, fourth instar day 0 larvae, and fourth instar molting larvae, the scaffold silk used to attach the cocoon to the substrate and the cocoon silk. Analysis by liquid chromatography-tandem mass spectrometry identified 500 proteins from the seven silks. In addition to the expected fibroins, sericins, and some known protease inhibitors, we also identified further protease inhibitors, enzymes, proteins of unknown function, and other proteins. Unsurprisingly, our quantitative results showed fibroins and sericins were the most abundant proteins in all seven silks. Except for fibroins and sericins, protease inhibitors, enzymes, and proteins of unknown function were more abundant than other proteins. We found significant change in silk protein compositions through development, being consistent with their different biological functions and complicated formation.

  14. The effects of BmNPV on biochemical changes in primary cultures of Bombyx mori embryonic tissue.

    PubMed

    Matindoost, Leila; Sendi, Jalal Jalali; Soleimanjahi, Hoorieh; Etebari, Kayvan; Rahbarizade, Fateme

    2008-01-01

    The effect of Bombyx mori nuclear polyhedrosis virus (BmNPV) on biochemical changes of TC-100 medium containing 10% fetal bovine serum (FBS) in embryonic primary cultures of silkworm was investigated. The primary cultures that reached 60% confluence were infected by 0.5, 1, and 2-ml viral inoculums (diluted with TC-100 medium representing multiplicity of infection (MOI) of 0.25, 0.5, and 1). Glucose, uric acid, urea, total protein, cholesterol, and alkaline phosphatase were measured in the medium of BmNPV-infected primary cultures. All biochemical compounds showed significant changes. Glucose decreased considerably by about 55 mg/ml, while different concentrations of the virus inoculums did not demonstrate significant differences among them. Total protein had only increased in 2 ml concentration and there were no changes in other concentrations. Uric acid as a by-product accumulated dramatically in all concentrations, while the amount of urea reduced in all treatments and this reduction was more evident in lower concentrations. Cholesterol consumption was high in cultures postinfection, while alkaline phosphatase (ALP) activity decreased in infected cells.

  15. Ricin-B-lectin enhances microsporidia Nosema bombycis infection in BmN cells from silkworm Bombyx mori.

    PubMed

    Liu, Han; Li, Mingqian; Cai, Shunfeng; He, Xinyi; Shao, Yongqi; Lu, Xingmeng

    2016-11-01

    Nosema bombycis is an obligate intracellular parasitic fungus that utilizes a distinctive mechanism to infect Bombyx mori Spore germination can be used for host cell invasion; however, the detailed mechanism remains to be elucidated. The ricin-B-lectin (RBL) gene is significantly differentially regulated after N. bombycis spore germination, and NbRBL might play roles in spore germination and infection. In this study, the biological function of NbRBL was examined. Protein sequence analysis showed that NbRBL is a secreted protein that attaches to carbohydrates. The relative expression level of the NbRBL gene was low during the first 30 h post-infection (hpi) in BmN cells, and high expression was detected from 42 hpi. Gene cloning, prokaryotic expression, and antibody preparation for NbRBL were performed. NbRBL was detected in total and secreted proteins using western blot analysis. Subcellular localization analysis showed that NbRBL is an intracellular protein. Spore adherence and infection assays showed that NbRBL could enhance spore adhesion to BmN cells; the proliferative activities of BmN cells incubated with anti-NbRBL were higher than those in negative control groups after N. bombycis infection; and the treatment groups showed less damage from spore invasion. We therefore, propose that NbRBL is released during spore germination, enhances spore adhesion to BmN cells, and contributes to spore invasion.

  16. Identification and characterization of two chitin-binding proteins from the peritrophic membrane of the silkworm, Bombyx mori L.

    PubMed

    Yang, Hua-Jun; Zhou, Fang; Malik, Firdose Ahmad; Bhaskar, Roy; Li, Xing-Hua; Hu, Jia-Biao; Sun, Chun-Guang; Miao, Yun-Gen

    2010-12-01

    The peritrophic membrane (PM) is a semi-permeable lining of the insect midgut, broadly analogous to the mucous lining of vertebrate gut. The PM proteins are important achievements for the function of the PM. In this study, two chitin-binding proteins (BmPM-P43 and BmPM-P41) from the PM of the silkworm, Bombyx mori, were identified and cloned. These proteins showed the molecular mass of 43 and 41 kDa, respectively. The deduced amino acid sequences codes for a protein of 381 amino acid residues and 364 amino acid residues, containing 12 and 14 cysteine residues followed by similar domain, both of them have 5 cysteine residues in similar position in the C-terminal. The confirmation of these proteins was performed by western blot analysis of recombinant BmPM-P43 and BmPM-P41. The chitin-binding activity analysis showed that the BmPM-P43 and BmPM-P41 could bind to chitin strongly. It is concluded that BmPM-P43 and BmPM-P41 contains a polysaccharide deacetylase domain instead of peritrophin domain, indicated that these two proteins may belong to a new chitin-binding protein family.

  17. Molecular Cloning, Bioinformatic Analysis, and Expression of Bombyx mori Lebocin 5 Gene Related to Beauveria bassiana Infection.

    PubMed

    Lü, Dingding; Hou, Chengxiang; Qin, Guangxing; Gao, Kun; Chen, Tian; Guo, Xijie

    2017-01-01

    A full-length cDNA of lebocin 5 (BmLeb5) was first cloned from silkworm, Bombyx mori, by rapid amplification of cDNA ends. The BmLeb5 gene is 808 bp in length and the open reading frame encodes a 179-amino acid hydroxyproline-rich peptide. Bioinformatic analysis results showed that BmLeb5 owns an O-glycosylation site and four RXXR motifs as other lebocins. Sequence similarity and phylogenic analysis results indicated that lebocins form a multiple gene family in silkworm as cecropins. Quantitative real-time PCR analysis revealed that BmLeb5 was highest expressed in the fat body. In the silkworm larvae infected by Beauveria bassiana, the expression level of BmLeb5 was upregulated in the fat body and hemolymph which are the most important immune tissues in silkworm. The recombinant protein of BmLeb5 was for the first time successfully expressed with prokaryotic expression system and purified. There are no reports so far that the expression of lebocins could be induced by entomopathogenic fungus. Our study suggested that BmLeb5 might play an important role in the immune response of silkworm to defend B. bassiana infection. The results also provided helpful information for further studying the lebocin family functioned in antifungal immune response in the silkworm.

  18. Chinese Folktales for Children.

    ERIC Educational Resources Information Center

    Kwok, Irene

    This bilingual text contains ten traditional Chinese folktales which have been rewritten for children. Each story deals with interpersonal relationships and/or stresses the Chinese way of life. Each page of text is given first in English and then in Chinese and is illustrated with a full-page drawing. The titles of the folktales are: (1) "One…

  19. Chinese Folktales for Children.

    ERIC Educational Resources Information Center

    Kwok, Irene

    This bilingual text contains ten traditional Chinese folktales which have been rewritten for children. Each story deals with interpersonal relationships and/or stresses the Chinese way of life. Each page of text is given first in English and then in Chinese and is illustrated with a full-page drawing. The titles of the folktales are: (1) "One…

  20. Computers and Chinese Linguistics.

    ERIC Educational Resources Information Center

    Kierman, Frank A.; Barber, Elizabeth

    This survey of the field of Chinese language computational linguistics was prepared as a background study for the Chinese Linguistics Project at Princeton. Since the authors' main purpose was "critical reconnaissance," quantitative emphasis is on systems with which they are most familiar. The complexity of the Chinese writing system has presented…

  1. MANDARIN CHINESE DICTIONARY.

    ERIC Educational Resources Information Center

    WANG, FRED FANGYU

    IN RESPONSE TO THE NEEDS OF THE GROWING NUMBER OF AMERICAN HIGH SCHOOL AND COLLEGE STUDENTS LEARNING CHINESE, SETON HALL UNIVERSITY UNDERTOOK A CONTRACT WITH THE U.S. OFFICE OF EDUCATION TO COMPILE A BILINGUAL POCKET-SIZE DICTIONARY FOR BEGINNING STUDENTS OF SPOKEN MANDARIN CHINESE. THE PRESENT WORK IS THE CHINESE TO ENGLISH SECTION IN PRELIMINARY…

  2. Hedgehog signaling pathway regulated the target genes for adipogenesis in silkworm Bombyx mori.

    PubMed

    Liang, Shuang; Chen, Rui-Ting; Zhang, Deng-Pan; Xin, Hu-Hu; Lu, Yan; Wang, Mei-Xian; Miao, Yun-Gen

    2015-10-01

    Hedgehog (Hh) signals regulate invertebrate and vertebrate development, yet the role of the pathway in adipose development remains poorly understood. In this report, we found that Hh pathway components are expressed in the fat body of silkworm larvae. Functional analysis of these components in a BmN cell line model revealed that activation of the Hh gene stimulated transcription of Hh pathway components, but inhibited the expression of the adipose marker gene AP2. Conversely, specific RNA interference-mediated knockdown of Hh resulted in increased AP2 expression. This further showed the regulation of Hh signal on the adipose marker gene. In silkworm larval models, enhanced adipocyte differentiation and an increase in adipocyte cell size were observed in silkworms that had been treated with a specific Hh signaling pathway antagonist, cyclopamine. The fat-body-specific Hh blockade tests were consistent with Hh signaling inhibiting silkworm adipogenesis. Our results indicate that the role of Hh signaling in inhibiting fat formation is conserved in vertebrates and invertebrates. © 2014 Institute of Zoology, Chinese Academy of Sciences.

  3. Proteomic analysis of BmN cells (Bombyx mori) in response to infection with Nosema bombycis.

    PubMed

    He, Xinyi; He, Xiangkang; Liu, Han; Li, Mingqian; Cai, Shunfeng; Fu, Zhangwuke; Lu, Xingmeng

    2014-11-01

    Nosema bombycis (N. bombycis, Nb) is an obligate intracellular parasite, which can cause pebrine disease in the silkworm. To investigate the effects of N. bombycis infection on the host cells, proteomes from BmN cells that had or had not been infected with N. bombycis at different infection stages were characterized with two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry, which identified 24 differentially expressed host proteins with significant intensity differences (P < 0.05) at least at one time point in mock- and N. bombycis infected cells. Notably, gene ontology analyses showed that these proteins are involved in many important biological reactions. During the infection phase, proteins involved in energy metabolism and oxidative stress had up-regulated expression. Two proteins participated in ubiquitin-dependent protein catabolic process had down-regulated expression. Quantitative real-time polymerase chain reaction was used to analyze the transcriptional profiles of these identified proteins. Taken together, the abundance changes, putative functions, and participation in biological reactions for the identified proteins produce a host-responsive protein model in N. bombycis-infected BmN cells. These findings further our knowledge about the effect of energy defect parasites on the host cells. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

  4. Traditional Chinese Medicine Induced Liver Injury

    PubMed Central

    2014-01-01

    Traditional Chinese Medicine (TCM) is popular around the world and encompasses many different practices with particular emphasis on herbal TCM. Using the PubMed database, a literature search was undertaken to assess the extent herbal TCM products exert rare hepatotoxicity. Analysis of reported cases revealed numerous specified herbal TCM products with potential hepatotoxicity. Among these were An Shu Ling, Bai Fang, Bai Xian Pi, Ban Tu Wan, Bo He, Bo Ye Qing Niu Dan, Bofu Tsu Sho San, Boh Gol Zhee, Cang Er Zi, Chai Hu, Chaso, Chi R Yun, Chuan Lian Zi, Ci Wu Jia, Da Chai Hu Tang, Da Huang, Du Huo, Gan Cao, Ge Gen, Ho Shou Wu, Hu Bohe You, Hu Zhang, Huang Qin, Huang Yao Zi, Hwang Geun Cho, Ji Gu Cao, Ji Ji, Ji Xue Cao, Jiguja, Jin Bu Huan, Jue Ming Zi, Kamishoyosan, Kudzu, Lei Gong Teng, Long Dan Xie Gan Tang, Lu Cha, Ma Huang, Mao Guo Tian Jie Cai, Onshido, Polygonum multiflorum, Qian Li Guang, Ren Shen, Sairei To, Shan Chi, Shen Min, Shi Can, Shi Liu Pi, Shou Wu Pian, Tian Hua Fen, White flood, Wu Bei Zi, Xi Shu, Xiao Chai Hu Tang, Yin Chen Hao, Zexie, Zhen Chu Cao, and various unclassified Chinese herbal mixtures. Causality was firmly established for a number of herbal TCM products by a positive reexposure test result, the liver specific scale of CIOMS (Council for International Organizations of Medical Sciences), or both. Otherwise, the quality of case data was mixed, especially regarding analysis of the herb ingredients because of adulteration with synthetic drugs, contamination with heavy metals, and misidentification. In addition, non-herbal TCM elements derived from Agaricus blazei, Agkistrodon, Antelope, Bombyx, Carp, Fish gallbladder, Phellinus, Scolopendra, Scorpio, and Zaocys are also known or potential hepatotoxins. For some patients, the clinical course was severe, with risks for acute liver failure, liver transplantation requirement, and lethality. In conclusion, the use of few herbal TCM products may rarely be associated with hepatotoxicity in some

  5. Nosema bombycis (Microsporidia) suppresses apoptosis in BmN cells (Bombyx mori).

    PubMed

    He, Xinyi; Fu, Zhangwuke; Li, Mingqian; Liu, Han; Cai, Shunfeng; Man, Nana; Lu, Xingmeng

    2015-09-01

    Nosema bombycis (N. bombycis, Nb) is a fungus-related and obligate intracellular parasite that causes chronic pebrine disease in the silkworm. After infecting the host, spores obtain energy from host cells and survive for several days. This symbiosis between the pathogen and the host cell suggests that N. bombycis prevents apoptosis and reactive oxygen species (ROS) production of host cells to create the optimal environmental conditions for its growth and development. In this study, different methods were used to prove that N. bombycis suppressed apoptosis in BmN cells. Flow cytometry analysis results showed that spores suppressed apoptosis of BmN cells at 2 and 5 days after infection (P < 0.05). Compared with actinomycin D (ActD) treatment, apoptosis of BmN cells was apparently reduced after spore infection (P < 0.01). Forty-eight hours after infection, the ROS production of BmN cells was down-regulated compared with that after ActD treatment for 6 h. Furthermore, N. bombycis prevented the formation of apoptosomes by down-regulating the expression of apaf-1 and cytochrome C. In addition, N. bombycis also up-regulated the expression of buffy. Western blot analysis demonstrated that spores decreased the level of host cytochrome C at 48 and 98 h post infection. Thus, our results suggested that N. bombycis inhibited the mitochondrial apoptotic pathway of the host cells to create an optimal environment for its own survival. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

  6. Transcriptome analysis of the brain of the silkworm Bombyx mori infected with Bombyx mori nucleopolyhedrovirus: A new insight into the molecular mechanism of enhanced locomotor activity induced by viral infection.

    PubMed

    Wang, Guobao; Zhang, Jianjia; Shen, Yunwang; Zheng, Qin; Feng, Min; Xiang, Xingwei; Wu, Xiaofeng

    2015-06-01

    Baculoviruses have been known to induce hyperactive behavior in their lepidopteran hosts for over a century. As a typical lepidopteran insect, the silkworm Bombyx mori displays enhanced locomotor activity (ELA) following infection with B. mori nucleopolyhedrovirus (BmNPV). Some investigations have focused on the molecular mechanisms underlying this abnormal hyperactive wandering behavior due to the virus; however, there are currently no reports about B. mori. Based on previous studies that have revealed that behavior is controlled by the central nervous system, the transcriptome profiles of the brains of BmNPV-infected and non-infected silkworm larvae were analyzed with the RNA-Seq technique to reveal the changes in the BmNPV-infected brain on the transcriptional level and to provide new clues regarding the molecular mechanisms that underlies BmNPV-induced ELA. Compared with the controls, a total of 742 differentially expressed genes (DEGs), including 218 up-regulated and 524 down-regulated candidates, were identified, of which 499, 117 and 144 DEGs could be classified into GO categories, KEGG pathways and COG annotations by GO, KEGG and COG analyses, respectively. We focused our attention on the DEGs that are involved in circadian rhythms, synaptic transmission and the serotonin receptor signaling pathway of B. mori. Our analyses suggested that these genes were related to the locomotor activity of B. mori via their essential roles in the regulations of a variety of behaviors and the down-regulation of their expressions following BmNPV infection. These results provide new insight into the molecular mechanisms of BmNPV-induced ELA.

  7. 20-Hydroxyecdysone-induced transcriptional activity of FoxO upregulates brummer and acid lipase-1 and promotes lipolysis in Bombyx fat body.

    PubMed

    Hossain, Muktadir S; Liu, Yan; Zhou, Shun; Li, Kang; Tian, Ling; Li, Sheng

    2013-09-01

    In a previous study, we have shown that the molting hormone, 20-hydroxyecdysone (20E), reduces insect food consumption resulting in fat body lipolysis during the non-feeding molting and pupation stages, and assumed that the transcription factor FoxO is involved in this process. To verify this hypothesis, we cloned foxO from the silkworm, Bombyx mori. During molting and pupation, FoxO is highly expressed and predominantly localizes in the nuclei of fat body cells. 20E induced foxO mRNA expression and FoxO nuclear localization resulting in an increase of FoxO transcriptional activity. RNAi of foxO prior to the 4th larval molting downregulated two lipase genes--the insect adipose triacylglycerol lipase homologue, brummer, and an acid lipase, acid lipase-1, in the fat body. Overexpression of the constitutively-active form of foxO (foxO(CA)) upregulated brummer and acid lipase-1 in both the fat body and Bombyx Bm-12 cells. Putative FoxO-response elements (FREs) are present in the promoter regions of brummer and acid lipase-1, and mutation of the FREs attenuated their FoxO-induced luciferase activities. ChIP assay revealed that FoxO binds directly to those FREs. Moreover, foxO(CA) overexpression in vivo doubled lipid concentration in the hemolymph, increased total lipase activity, and slightly but significantly reduced lipid content in the fat body. Taken together, we conclude that 20E increases the transcriptional activity of FoxO which, in turn, upregulates brummer and acid lipase-1 and induces lipolysis in the Bombyx fat body during molting and pupation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Functional analysis of sex-determination genes by gene silencing with LNA-DNA gapmers in the silkworm, Bombyx mori.

    PubMed

    Sakai, Hiroki; Sakaguchi, Honami; Aoki, Fugaku; Suzuki, Masataka G

    2015-08-01

    The sexual fate of B. mori is determined genetically; ZW, female and ZZ, male. Recently, we successfully identified a strong candidate gene at the top of the sex determination cascade in B. mori. This gene was termed Feminizer (Fem) and revealed to be a source of Fem-piRNA. Further, we found that B. mori doublesex (Bmdsx) splicing was markedly altered to produce the male-type isoform when a Fem-piRNA inhibitor was injected into ZW embryos. Moreover, knockdown of Masculinizer (Masc), a Fem-piRNA target gene, altered to produce the female-type isoform of Bmdsx in male embryos. However, it remains unclear as to whether Masc directly regulates the sex-specific expression of Bmdsx. In previous studies, we determined that the male-specific isoform of the Bombyx homolog of IGF-II mRNA-binding protein (Imp(M)) was involved in the male-specific splicing of Bmdsx. In an attempt to clarify the genetic relationship between Fem, Masc, Imp(M), and Bmdsx, knockdown experiments were performed. Knockdown of Fem shifted into male-type Bmdsx, Imp(M) and Masc in female embryos. Knockdown of Masc led to the production of the female-type Bmdsx and a dramatic reduction in Imp(M) expression in male embryos. Knockdown of Imp(M) shifted Bmdsx splice mode from the male-type into the female-type. Our results suggest that: (1) Fem reduces Masc expression, (2) Masc dramatically induces Imp(M) expression, and (3) Imp(M) shifting Bmdsx splice mode from the female-type into the male-type. Based on these findings, we propose a possible genetic cascade regulating sex determination in B. mori.

  9. A genome-wide survey for host response of silkworm, Bombyx mori during pathogen Bacillus bombyseptieus infection.

    PubMed

    Huang, Lulin; Cheng, Tingcai; Xu, Pingzhen; Cheng, Daojun; Fang, Ting; Xia, Qingyou

    2009-12-01

    Host-pathogen interactions are complex relationships, and a central challenge is to reveal the interactions between pathogens and their hosts. Bacillus bombysepticus (Bb) which can produces spores and parasporal crystals was firstly separated from the corpses of the infected silkworms (Bombyx mori). Bb naturally infects the silkworm can cause an acute fuliginosa septicaemia and kill the silkworm larvae generally within one day in the hot and humid season. Bb pathogen of the silkworm can be used for investigating the host responses after the infection. Gene expression profiling during four time-points of silkworm whole larvae after Bb infection was performed to gain insight into the mechanism of Bb-associated host whole body effect. Genome-wide survey of the host genes demonstrated many genes and pathways modulated after the infection. GO analysis of the induced genes indicated that their functions could be divided into 14 categories. KEGG pathway analysis identified that six types of basal metabolic pathway were regulated, including genetic information processing and transcription, carbohydrate metabolism, amino acid and nitrogen metabolism, nucleotide metabolism, metabolism of cofactors and vitamins, and xenobiotic biodegradation and metabolism. Similar to Bacillus thuringiensis (Bt), Bb can also induce a silkworm poisoning-related response. In this process, genes encoding midgut peritrophic membrane proteins, aminopeptidase N receptors and sodium/calcium exchange protein showed modulation. For the first time, we found that Bb induced a lot of genes involved in juvenile hormone synthesis and metabolism pathway upregulated. Bb also triggered the host immune responses, including cellular immune response and serine protease cascade melanization response. Real time PCR analysis showed that Bb can induce the silkworm systemic immune response, mainly by the Toll pathway. Anti-microorganism peptides (AMPs), including of Attacin, Lebocin, Enbocin, Gloverin and Moricin

  10. Genome-wide identification and immune response analysis of serine protease inhibitor genes in the silkworm, Bombyx mori.

    PubMed

    Zhao, Ping; Dong, Zhaoming; Duan, Jun; Wang, Genhong; Wang, Lingyan; Li, Youshan; Xiang, Zhonghuai; Xia, Qingyou

    2012-01-01

    In most insect species, a variety of serine protease inhibitors (SPIs) have been found in multiple tissues, including integument, gonad, salivary gland, and hemolymph, and are required for preventing unwanted proteolysis. These SPIs belong to different families and have distinct inhibitory mechanisms. Herein, we predicted and characterized potential SPI genes based on the genome sequences of silkworm, Bombyx mori. As a result, a total of eighty SPI genes were identified in B. mori. These SPI genes contain 10 kinds of SPI domains, including serpin, Kunitz_BPTI, Kazal, TIL, amfpi, Bowman-Birk, Antistasin, WAP, Pacifastin, and alpha-macroglobulin. Sixty-three SPIs contain single SPI domain while the others have at least two inhibitor units. Some SPIs also contain non-inhibitor domains for protein-protein interactions, including EGF, ADAM_spacer, spondin_N, reeler, TSP_1 and other modules. Microarray analysis showed that fourteen SPI genes from lineage-specific TIL family and Group F of serpin family had enriched expression in the silk gland. The roles of SPIs in resisting pathogens were investigated in silkworms when they were infected by four pathogens. Microarray and qRT-PCR experiments revealed obvious up-regulation of 8, 4, 3 and 3 SPI genes after infection with Escherichia coli, Bacillus bombysepticus, Beauveria bassiana or B. mori nuclear polyhedrosis virus (BmNPV), respectively. On the contrary, 4, 11, 7 and 9 SPI genes were down-regulated after infection with E. coli, B. bombysepticus, B. bassiana or BmNPV, respectively. These results suggested that these SPI genes may be involved in resistance to pathogenic microorganisms. These findings may provide valuable information for further clarifying the roles of SPIs in the development, immune defence, and efficient synthesis of silk gland protein.

  11. A single sex pheromone receptor determines chemical response specificity of sexual behavior in the silkmoth Bombyx mori.

    PubMed

    Sakurai, Takeshi; Mitsuno, Hidefumi; Haupt, Stephan Shuichi; Uchino, Keiro; Yokohari, Fumio; Nishioka, Takaaki; Kobayashi, Isao; Sezutsu, Hideki; Tamura, Toshiki; Kanzaki, Ryohei

    2011-06-01

    In insects and other animals, intraspecific communication between individuals of the opposite sex is mediated in part by chemical signals called sex pheromones. In most moth species, male moths rely heavily on species-specific sex pheromones emitted by female moths to identify and orient towards an appropriate mating partner among a large number of sympatric insect species. The silkmoth, Bombyx mori, utilizes the simplest possible pheromone system, in which a single pheromone component, (E, Z)-10,12-hexadecadienol (bombykol), is sufficient to elicit full sexual behavior. We have previously shown that the sex pheromone receptor BmOR1 mediates specific detection of bombykol in the antennae of male silkmoths. However, it is unclear whether the sex pheromone receptor is the minimally sufficient determination factor that triggers initiation of orientation behavior towards a potential mate. Using transgenic silkmoths expressing the sex pheromone receptor PxOR1 of the diamondback moth Plutella xylostella in BmOR1-expressing neurons, we show that the selectivity of the sex pheromone receptor determines the chemical response specificity of sexual behavior in the silkmoth. Bombykol receptor neurons expressing PxOR1 responded to its specific ligand, (Z)-11-hexadecenal (Z11-16:Ald), in a dose-dependent manner. Male moths expressing PxOR1 exhibited typical pheromone orientation behavior and copulation attempts in response to Z11-16:Ald and to females of P. xylostella. Transformation of the bombykol receptor neurons had no effect on their projections in the antennal lobe. These results indicate that activation of bombykol receptor neurons alone is sufficient to trigger full sexual behavior. Thus, a single gene defines behavioral selectivity in sex pheromone communication in the silkmoth. Our findings show that a single molecular determinant can not only function as a modulator of behavior but also as an all-or-nothing initiator of a complex species-specific behavioral sequence.

  12. Integrin beta and receptor for activated protein kinase C are involved in the cell entry of Bombyx mori cypovirus.

    PubMed

    Zhang, Yiling; Cao, Guangli; Zhu, Liyuan; Chen, Fei; Zar, Mian Sahib; Wang, Simei; Hu, Xiaolong; Wei, Yuhong; Xue, Renyu; Gong, Chengliang

    2017-02-07

    Receptor-mediated endocytosis using a β1 integrin-dependent internalization was considered as the primary mechanism for the initiation of mammalian reovirus infection. Bombyx mori cypovirus (BmCPV) is a member of Reoviridae family which mainly infects the midgut epithelium of silkworm; the cell entry of BmCPV is poorly explored. In this study, co-immunoprecipitation (Co-IP), virus overlay protein binding assay (VOPBA), and BmCPV-protein interaction on the polyvinylidene difluoride membrane (BmCPV-PI-PVDF) methods were employed to screen the interacting proteins of BmCPV, and several proteins including integrin beta and receptor for activated protein kinase C (RACK1) were identified as the candidate interacting proteins for establishing the infection of BmCPV. The infectivity of BmCPV was investigated in vivo and in vitro by RNA interference (RNAi) and antibody blocking methods, and the results showed that the infectivity of BmCPV was significantly reduced by either small interfering RNA-mediated silencing of integrin beta and RACK1 or antibody blocking of integrin beta and RACK1. The expression level of integrin beta or RACK1 is not the highest in the silkworm midgut which is a principal target tissue of BmCPV, suggesting that the molecules other than integrin beta or RACK1 might play a key role in determining the tissue tropism of BmCPV infection. The establishment of BmCPV infection depends on other factors, and these factors interacted with integrin beta and RACK1 to form receptor complex for the cell entry of BmCPV.

  13. Rheology and dynamic light scattering of silk fibroin solution extracted from the middle division of Bombyx mori silkworm.

    PubMed

    Ochi, Akie; Hossain, Khandker S; Magoshi, Jun; Nemoto, Norio

    2002-01-01

    Dynamic light scattering (DLS) and rheological measurements were performed on aqueous silk fibroin solutions extracted from the middle division of Bombyx mori silkworm over a wide range of polymer concentration C from 0.08 to 27.5 wt %. DLS results obtained in the dilute region of C less than 1 wt % are consistent with a model that an elementary unit is a large protein complex consisting of silk fibroin and P25 with a 6:1 molar ratio. Rheological measurements in the dilute C region reveal that those units (or clusters) with the hydrodynamic radius of about 100 nm form a network extending over the whole sample volume with small pseudoplateau modulus mainly by ionic bonding between COO(-) ions of the fibroin molecules and divalent metallic ions such as Ca(2+) or Mg(2+) ions present in the sample and also that, after a yield stress is reached, steady plastic flow is induced with viscosity much lower than the zero-shear viscosity estimated from creep and creep recovery measurements by 4-6 orders of magnitude. Angular frequency omega dependencies of the storage and the loss shear moduli, G'(omega) and G' '(omega), measured in the linear viscoelastic region, indicate that all solutions possess the pseudoplateau modulus in the low omega region and samples become highly viscoleastic for C greater, similar 4.2 wt %. Above C = 11.2 wt % another plateau appears at the high omega end accompanied by a distinct maximum of G' ' in the intermediate omega region. The relaxation motion with tau = 0.5 s corresponding to the maximum of G' ' is one of characteristic properties of the fibroin solutions in the high C region. Thermorheological behaviors of the solution with C = 27.5 wt % show that the network structure formed in the MM part of the silk gland is susceptible to temperature and a more stable homogeneous network is realized by raising the temperature up to T = 65 degrees C.

  14. Scientific validation of cardioprotective attribute by standardized extract of Bombyx mori against doxorubicin-induced cardiotoxicity in murine model

    PubMed Central

    Khan, Masood S.; Singh, Mhaveer; Khan, Mohammad A.; Arya, D. S.; Ahmad, Sayeed

    2014-01-01

    Doxorubicin (DOX) is an excellent antineoplastic agent used for the treatment of hematological and solid malignancies. The aqueous extract of Bombyx mori (BMAE) contains amino acids and some flavonoids with obvious cardioprotective effect. The aim of this study was to investigate the possible protective effect of BMAE against DOX-induced cardiotoxicity and its underlying mechanisms on murine model. The metabolic profiling of BMAE was carried out by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) and the amino acid profiling by HPLC method using fluorescence detector (HPLC-FLD). The biochemical parameter like caspase-3, tumor necrosis factor–alpha (TNF-α), interleukin -6 (IL-6), creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) and malondialdehyde (MDA) were studied. Tissue damage was further evaluated by histopathological studies. The metabolic profiling of BMAE exhibited presence of quercetin 7-O-ß-D-glucoside, kaempferol 7-O-ß-D-glucopyranoside, coumaric acid glucoside, 2-hydroxy-nonadecanoic acid and 9,12-dihydroxy stearic acid as important constituents. The amino acid profile by HPLC-FLD showed presence of 17 amino acids. The BMAE showed prominent free radical scavenging activity when assessed by the H2O2 and super-oxide method. The results of present investigation showed protection against DOX-induced oxidative stress (lipid peroxidation), by reverting activities of apoptotic markers (caspase-3 and TNF-α), cardiac markers (CK-MB and LDH activities) as well as pro-inflammatory marker IL-6 followed by oral administration of BMAE. In addition, results of histopathology also supported well the above results. It was observed that BMAE protects DOX-induced cardiotoxicity by virtue of its antioxidants possibly by flavonoids and amino acids. PMID:26417320

  15. Tuning Molecular Weights of Bombyx mori (B. mori) Silk Sericin to Modify Its Assembly Structures and Materials Formation

    PubMed Central